Oxime derivative substituted hydroxyethylamine aspartyl protease inhibitors

ABSTRACT

The invention relates to novel compounds and methods of treating diseases, disorders, and conditions associated with amyloidosis. Amyloidosis refers to a collection of diseases, disorders, and conditions associated with abnormal deposition of A-beta protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority under 35 U.S.C. § 119(e)to U.S. Provisional Application 60/586,207, filed Jul. 9, 2004, U.S.Provisional Application 60/608,143, filed Sep. 9, 2004, U.S. ProvisionalApplication 60/656,873, filed Mar. 1, 2005, and U.S. ProvisionalApplication 60/681,138, filed May 16, 2005, incorporated herein byreference in full.

FIELD OF THE PRESENT INVENTION

The present invention is directed to novel compounds and also to methodsof treating at least one condition, disorder, or disease associated withamyloidosis using such compounds.

BACKGROUND OF THE PRESENT INVENTION

Amyloidosis refers to a collection of conditions, disorders, anddiseases associated with abnormal deposition of amyloidal protein. Forinstance, Alzheimer's disease is believed to be caused by abnormaldeposition of amyloidal protein in the brain. Thus, these amyloidalprotein deposits, otherwise known as amyloid-beta peptide, A-beta, orbetaA4, are the result of proteolytic cleavage of the amyloid precursorprotein (APP).

The majority of APP molecules that undergo proteolytic cleavage arecleaved by the aspartyl protease alpha-secretase. Alpha-secretasecleaves APP between Lys687 and Leu688 producing a large, solublefragment, alpha-sAPP, which is a secreted form of APP that does notresult in beta-amyloid plaque formation. The alpha-secretase cleavagepathway precludes the formation of A-beta, thus providing an alternatetarget for preventing or treating amyloidosis.

Some APP molecules, however, are cleaved by a different aspartylprotease known as beta-secretase which is also referred to in theliterature as BACE, BACE1, Asp2, and Memapsin2. Beta-secretase cleavesAPP after Met671, creating a C-terminal fragment. See, for example,Sinha et al., Nature, (1999), 402:537-554 and published PCT applicationWO 00/17369. After cleavage of APP by beta-secretase, an additionalaspartyl protease, gamma-secretase, may then cleave the C-terminus ofthis fragment, at either Val711 or Ile713, (found within the APPtransmembrane domain), generating an A-beta peptide. The A-beta peptidemay then proceed to form beta-amyloid plaques. A detailed description ofthe proteolytic processing of APP fragments is found, for example, inU.S. Pat. Nos. 5,441,870, 5,721,130, and 5,942,400.

The amyloidal disease Alzheimer's is a progressive degenerative diseasethat is characterized by two major pathologic observations in the brainwhich are (1) neurofibrillary tangles, and (2) beta-amyloid (orneuritic) plaques. A major factor in the development of Alzheimer'sdisease is A-beta deposits in regions of the brain responsible forcognitive activities. These regions include, for example, thehippocampus and cerebral cortex. A-beta is a neurotoxin that may becausally related to neuronal death observed in Alzheimer's diseasepatients. See, for example, Selkoe, Neuron, 6 (1991) 487. Since A-betapeptide accumulates as a result of APP processing by beta-secretase,inhibiting beta-secretase's activity is desirable for the treatment ofAlzheimer's disease.

Dementia-characterized disorders also arise from A-beta accumulation inthe brain including accumulation in cerebral blood vessels (known asvasculary amyloid angiopathy) such as in the walls of meningeal andparenchymal arterioles, small arteries, capillaries, and venules. A-betamay also be found in cerebrospinal fluid of both individuals with andwithout Alzheimer's disease. Additionally, neurofibrillary tanglessimilar to the ones observed in Alzheimer's patients can also be foundin individuals without Alzheimer's disease. In this regard, a patientexhibiting symptoms of Alzheimer's due to A-beta deposits andneurofibrillary tangles in their cerebrospinal fluid may in fact besuffering from some other form of dementia. See, for example, Seubert etal., Nature, 359 (1992) 325-327. Examples of other forms of dementiawhere A-beta accumulation generates amyloidogenic plaques or results invascular amyloid angiopathy include Trisomy 21 (Down's Syndrome),Hereditary Cerebral Hemorrhage with amyloidosis of the Dutch-Type(HCHWA-D), and other neurodegenerative disorders. Consequently,inhibiting beta-secretase is not only desirable for the treatment ofAlzheimer's, but also for the treatment of other conditions associatedwith amyloidosis.

Amyloidosis is also implicated in the pathophysiology of stroke.Cerebral amyloid angiopathy is a common feature of the brains of strokepatients exhibiting symptoms of dementia, focal neurological syndromes,or other signs of brain damage. See, for example, Corio et al.,Neuropath Appl. Neurobiol., 22 (1996) 216-227. This suggests thatproduction and deposition of A-beta may contribute to the pathology ofAlzheimer's disease, stroke, and other diseases and conditionsassociated with amyloidosis. Accordingly, the inhibition of A-betaproduction is desirable for the treatment of Alzheimer's disease,stroke, and other diseases and conditions associated with amyloidosis.

Presently there are no known effective treatments for preventing,delaying, halting, or reversing the progression of Alzheimer's diseaseand other conditions associated with amyloidosis. Consequently, there isan urgent need for methods of treatment capable of preventing andtreating conditions associated with amyloidosis including Alzheimer'sdisease.

Likewise, there is a need for methods of treatment using compounds thatinhibit beta-secretase-mediated cleavage of APP. There is also a needfor methods of treatment using compounds that are effective inhibitorsof A-beta production, and/or are effective at reducing A-beta depositsor plaques, as well as methods of treatment capable of combatingdiseases and conditions characterized by amyloidosis, or A-betadeposits, or plaques.

There is also a need for methods of treating conditions associated withamyloidosis using compounds that are efficacious, bioavailable and/orselective for beta-secretase. An increase in efficacy, selectivity,and/or oral bioavailability may result in preferred, safer, lessexpensive products that are easier for patients to use.

There is also a need for methods of treating at least one conditionassociated with amyloidosis using compounds with characteristics thatwould allow them to cross the blood-brain-barrier. Desirablecharacteristics include a low molecular weight and a high log P(increased log P=increased lipophilicity).

Generally, known aspartyl protease inhibitors are either incapable ofcrossing the blood-brain barrier or do so with great difficulty. Thesecompounds are unsuitable for the treatment of the conditions describedherein. Accordingly, there is a need for methods of treating at leastone condition associated with amyloidosis using compounds that canreadily cross the blood-brain barrier and inhibit beta-secretase.

There is also a need for a method of finding suitable compounds forinhibiting beta-secretase activity, inhibiting cleavage of APP,inhibiting production of A-beta, and/or reducing A-beta deposits orplaques.

The present invention is directed to novel compounds and also to methodsof treating at least one condition, disorder, or disease associated withamyloidosis using such compounds. An embodiment of the present inventionis compounds of formula (I) or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are defined below. Anotherembodiment of the present invention is a method of administering atleast one compound of formula (I) or at least one pharmaceuticallyacceptable salt thereof, wherein R₁, R₂, and R_(C) are defined below, intreating at least one condition, disorder, or disease associated withamyloidosis. Another embodiment is directed to methods of treatmentcomprising administering at least one compound of formula (I) or atleast one pharmaceutically acceptable salt thereof, wherein R₁, R₂, andR_(C) are defined below, useful in preventing, delaying, halting, orreversing the progression of Alzheimer's disease.

Another embodiment of the present invention is directed to uses ofbeta-secretase inhibitors of at least one compound of formula (I) or atleast one pharmaceutically acceptable salt thereof, wherein R₁, R₂, andR_(C) are defined below, in treating or preventing at least onecondition, disorder, or disease associated with amyloidosis.

Another embodiment of the present invention is the administration ofbeta-secretase inhibitors of at least one compound of formula (I) or atleast one pharmaceutically acceptable salt thereof, wherein R₁, R₂, andR_(C) are defined below, exhibiting at least one property chosen fromimproved efficacy, bioavailability, selectivity, and blood-brain barrierpenetrating properties. The present invention accomplishes one or moreof these objectives and provides further related advantages.

BRIEF SUMMARY OF THE PRESENT INVENTION

The present invention is directed to novel compounds and also to methodsof treating at least one condition, disorder, or disease associated withamyloidosis using such compounds. The present invention is directed tocompounds of formula (I) or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are defined below, and methodsof treating at least one condition, disorder, or disease associated withamyloidosis. As previously noted, amyloidosis refers to a collection ofdiseases, disorders, and conditions associated with abnormal depositionof A-beta protein.

An embodiment of the present invention is to provide compounds havingproperties contributing to viable pharmaceutical compositions. Theseproperties include improved efficacy, bioavailability, selectivity,and/or blood-brain barrier penetrating properties. They can beinter-related, though an increase in any one of them correlates to abenefit for the compound and its corresponding method of treatment. Forexample, an increase in any one of these properties may result inpreferred, safer, less expensive products that are easier for patientsto use.

Accordingly, an embodiment of the present invention is to providecompounds of formula (I),

or pharmaceutically acceptable salts thereof, wherein R₁, R₂, and R_(C)are defined below.

Another embodiment of the present invention is a method of preventing ortreating at least one condition that benefits from inhibition of atleast one aspartyl-protease, comprising administering to a host acomposition comprising a therapeutically effective amount of at leastone compound of formula (I),

or at least one pharmaceutically acceptable salt thereof, wherein R₁,R₂, and R_(C) are defined below.

Another embodiment is to provide selective compounds of formula (I),

or pharmaceutically acceptable salts thereof, wherein R₁, R₂, and R_(C)are defined below.

Another embodiment is to provide efficacious compounds of formula (I),

or pharmaceutically acceptable salts thereof, wherein the inhibition isat least 10% for a dose of about 100 mg/kg or less, and wherein R₁, R₂,and R_(C) are defined below.

Another embodiment is to provide orally bioavailable compounds offormula (I),

or pharmaceutically acceptable salts thereof, wherein said compound hasan F value of at least 10%, and wherein R₁, R₂, and R_(C) are definedbelow.

Another embodiment of the present invention provides a method forpreventing or treating at least one condition that benefits frominhibition of at least one aspartyl-protease, comprising administeringto a host at least one compound of formula (I), or pharmaceuticallyacceptable salts thereof, wherein the inhibition is at least 10% for adose of 100 mg/kg or less, and wherein R₁, R₂, and R_(C) are definedbelow.

Another embodiment of the present invention provides a method forpreventing or treating at least one condition that benefits frominhibition of at least one aspartyl-protease, comprising administeringto a host a composition comprising a therapeutically effective amount ofat least one compound of formula (I), or pharmaceutically acceptablesalts thereof, wherein R₁, R₂, and R_(C) are as defined below.

Another embodiment of the present invention provides a method ofpreventing or treating at least one condition that benefits frominhibition of at least one aspartyl-protease, comprising administeringto a host a composition comprising a therapeutically effective amount ofat least one compound of formula (I), or pharmaceutically acceptablesalts thereof, wherein the inhibition is at least 10% for a dose of 100mg/kg or less, and wherein R₁, R₂, and R_(C) are as defined below.

Another embodiment provides a method of preventing or treating at leastone condition that benefits from inhibition of beta-secretase,comprising administering to a host a composition comprising atherapeutically effective amount of at least one compound of formula(I), or pharmaceutically acceptable salts thereof, wherein theinhibition is at least 10% for a dose of 100 mg/kg or less, and whereinR₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method forpreventing or treating at least one condition associated withamyloidosis, comprising administering to a patient in need thereof atherapeutically effective amount of at least one compound of formula(I), or at least one pharmaceutically acceptable salt thereof, thecompound having an F value of at least 10%, wherein R₁, R₂, and R_(C)are as defined below.

In another embodiment, the present invention provides a method ofpreventing or treating at least one condition associated withamyloidosis, comprising administering to a host a composition comprisinga therapeutically effective amount of at least one selectivebeta-secretase inhibitor of formula (I), or pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofpreventing or treating Alzheimer's disease by administering to a host aneffective amount of at least one compound of formula (I), or at leastone pharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C)are as defined below.

In another embodiment, the present invention provides a method ofpreventing or treating dementia by administering to a host an effectiveamount of at least one compound of formula (I), or pharmaceuticallyacceptable salt thereof, wherein R₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofinhibiting beta-secretase activity in a host, the method comprisingadministering to the host an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofinhibiting beta-secretase activity in a cell, the method comprisingadministering to the cell an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofinhibiting beta-secretase activity in a host, the method comprisingadministering to the host an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein the host is a human, and wherein R₁, R₂, and R_(C) areas defined below.

In another embodiment, the present invention provides a method ofaffecting beta-secretase-mediated cleavage of amyloid precursor proteinin a patient, comprising administering a therapeutically effectiveamount of at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C) areas defined below.

In another embodiment, the present invention provides a method ofinhibiting cleavage of amyloid precursor protein at a site betweenMet596 and Asp597 (numbered for the APP-695 amino acid isotype), or at acorresponding site of an isotype or mutant thereof, comprisingadministering a therapeutically effective amount of at least onecompound of formula (I), or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofinhibiting production of A-beta, comprising administering to a patient atherapeutically effective amount of at least one compound of formula(I), or at least one pharmaceutically acceptable salt thereof, whereinR₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofpreventing or treating deposition of A-beta, comprising administering atherapeutically effective amount of at least one compound of formula(I), or at least one pharmaceutically acceptable salt thereof, whereinR₁, R₂, and R_(C) are as defined below.

In another embodiment, the present invention provides a method ofpreventing, delaying, halting, or reversing a disease characterized byA-beta deposits or plaques, comprising administering a therapeuticallyeffective amount of at least one compound of formula (I), or at leastone pharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C)are as defined below.

In another embodiment, the A-beta deposits or plaques are in a humanbrain.

In another embodiment, the present invention provides a method ofinhibiting the activity of at least one aspartyl protease in a patientin need thereof, comprising administering a therapeutically effectiveamount of at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C) areas defined below.

In another embodiment, the at least one aspartyl protease isbeta-secretase.

In another embodiment, the present invention provides a method ofinteracting an inhibitor with beta-secretase, comprising administeringto a patient in need thereof a therapeutically effective amount of atleast one compound of formula (I), or at least one pharmaceuticallyacceptable salt thereof, wherein R₁, R₂, and R_(C) are as defined below,wherein the at least one compound interacts with at least onebeta-secretase subsite such as S1, S1′, or S2′.

In another embodiment, the present invention provides an article ofmanufacture, comprising (a) at least one dosage form of at least onecompound of formula (I), or pharmaceutically acceptable salt thereof,wherein R₁, R₂, and R_(C) are defined below, (b) a package insertproviding that a dosage form comprising a compound of formula (I) shouldbe administered to a patient in need of therapy for at least onedisorder, condition or disease associated with amyloidosis, and (c) atleast one container in which at least one dosage form of at least onecompound of formula (I) is stored.

In another embodiment, the present invention provides a packagedpharmaceutical composition for treating at least one condition relatedto amyloidosis, comprising (a) a container which holds an effectiveamount of at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C) areas defined below, and (b) instructions for using the pharmaceuticalcomposition.

DEFINTIONS

Throughout the specification and claims, including the detaileddescription below, the following definitions apply.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

Where multiple substituents are indicated as being attached to astructure, it is to be understood that the substituents can be the sameor different.

APP, amyloid precursor protein, is defined as any APP polypeptide,including APP variants, mutations, and isoforms, for example, asdisclosed in U.S. Pat. No. 5,766,846.

Beta-amyloid peptide (A-beta peptide) is defined as any peptideresulting from beta-secretase mediated cleavage of APP, including, forexample, peptides of 39, 40, 41, 42, and 43 amino acids, and extendingfrom the beta-secretase cleavage site to amino acids 39, 40, 41, 42, or43.

Beta-secretase is an aspartyl protease that mediates cleavage of APP atthe N-terminus edge of A-beta. Human beta-secretase is described, forexample, in WO 00/17369.

The term “complex” as used herein refers to an inhibitor-enzyme complex,wherein the inhibitor is a compound of formula (I) described herein andwherein the enzyme is beta-secretase or a fragment thereof.

The term “host” as used herein refers to a cell or tissue, in vitro orin vivo, an animal, or a human.

The term “treating” refers to administering a compound or a compositionof formula (I) to a host having at least a tentative diagnosis ofdisease or condition. The methods of treatment and compounds of thepresent invention will delay, halt, or reverse the progression of thedisease or condition thereby giving the host a longer and/or morefunctional life span.

The term “preventing” refers to administering a compound or acomposition of formula (I) to a host who has not been diagnosed ashaving the disease or condition at the time of administration, but whocould be expected to develop the disease or condition or be at increasedrisk for the disease or condition. The methods of treatment andcompounds of the present invention may slow the development of diseasesymptoms, delay the onset of the disease or condition, halt theprogression of disease development, or prevent the host from developingthe disease or condition at all. Preventing also includes administrationof at least one compound or a composition of the present invention tothose hosts thought to be predisposed to the disease or condition due toage, familial history, genetic or chromosomal abnormalities, due to thepresence of one or more biological markers for the disease or condition,such as a known genetic mutation of APP or APP cleavage products inbrain tissues or fluids, and/or due to environmental factors.

The term “halogen” in the present invention refers to fluorine, bromine,chlorine, or iodine.

The term “alkyl” in the present invention refers to straight or branchedchain alkyl groups having 1 to 20 carbon atoms. An alkyl group mayoptionally comprise at least one double bond and/or at least one triplebond. The alkyl groups herein are unsubstituted or substituted in one ormore positions with various groups. For example, such alkyl groups maybe optionally substituted with at least one group independently selectedfrom alkyl, alkoxy, —C(O)H, carboxy, alkoxycarbonyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, amido, alkanoylamino, amidino,alkoxycarbonylamino, N-alkyl amidino, N-alkyl amido, N,N′-dialkylamido,aralkoxycarbonylamino, halogen, alkyl thio, alkylsulfinyl,alkylsulfonyl, hydroxy, cyano, nitro, amino, monoalkylamino,dialkylamino, haloalkyl, haloalkoxy, aminoalkyl, monoalkylaminoalkyl,dialkylaminoalkyl, and the like. Additionally, at least one carbonwithin any such alkyl may be optionally replaced with —C(O)—.

Examples of alkyls include methyl, ethyl, ethenyl, ethynyl, propyl,1-ethyl-propyl, propenyl, propynyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, 2-methylbutyl, 3-methyl-butyl, 1-but-3-enyl,butynyl, pentyl, 2-pentyl, isopentyl, neopentyl, 3-methylpentyl,1-pent-3-enyl, 1-pent-4-enyl, pentyn-2-yl, hexyl, 2-hexyl, 3-hexyl,1-hex-5-enyl, formyl, acetyl, acetylamino, trifluoromethyl, propionicacid ethyl ester, trifluoroacetyl, methylsulfonyl, ethylsulfonyl,1-hydroxy-1-methylethyl, 2-hydroxy-1,1-dimethyl-ethyl,1,1-dimethyl-propyl, cyano-dimethyl-methyl, propylamino, and the like.

In an embodiment, alkyls may be selected from sec-butyl, isobutyl,ethynyl, 1-ethyl-propyl, pentyl, 3-methyl-butyl, pent-4-enyl, isopropyl,tert-butyl, 2-methylbutane, and the like.

In another embodiment, alkyls may be selected from formyl, acetyl,acetylamino, trifluoromethyl, propionic acid ethyl ester,trifluoroacetyl, methylsulfonyl, ethylsulfonyl, 1-hydroxy-1-methylethyl,2-hydroxy-1,1,-dimethyl-ethyl, 1,1-dimethyl-propyl,cyano-dimethyl-methyl, propylamino, and the like.

The term “alkoxy” in the present invention refers to straight orbranched chain alkyl groups, wherein an alkyl group is as defined above,and having 1 to 20 carbon atoms, attached through at least one divalentoxygen atom, such as, for example, methoxy, ethoxy, propoxy, isopropoxy,n-butoxy, sec-butoxy, tert-butoxy, pentoxy, isopentoxy, neopentoxy,hexyloxy, heptyloxy, allyloxy, 2-(2-methoxy-ethoxy)-ethoxy, benzyloxy,3-methylpentoxy, and the like.

In an embodiment, alkoxy groups may be selected from allyloxy, hexyloxy,heptyloxy, 2-(2-methoxy-ethoxy)-ethoxy, benzyloxy, and the like.

The term “—C(O)-alkyl” or “alkanoyl” refers to an acyl group derivedfrom an alkylcarboxylic acid, a cycloalkylcarboxylic acid, aheterocycloalkylcarboxylic acid, an arylcarboxylic acid, anarylalkylcarboxylic acid, a heteroarylcarboxylic acid, or aheteroarylalkylcarboxylic acid, examples of which include formyl,acetyl, 2,2,2-trifluoroacetyl, propionyl, butyryl, valeryl,4-methylvaleryl, and the like.

The term “cycloalkyl” refers to an optionally substituted carbocyclicring system of one or more 3, 4, 5, 6, 7, or 8 membered rings, including9, 10, 11, 12, 13, and 14 membered fused ring systems, all of which canbe saturated or partially unsaturated. The cycloalkyl may be monocyclic,bicyclic, tricyclic, and the like. Bicyclic and tricyclic as used hereinare intended to include both fused ring systems, such as adamantyl,octahydroindenyl, decahydro-naphthyl, and the like, substituted ringsystems, such as cyclopentylcyclohexyl, and spirocycloalkyls such asspiro[2.5]octane, spiro[4.5]decane, 1,4-dioxa-spiro[4.5]decane, and thelike. A cycloalkyl may optionally be a benzo fused ring system, which isoptionally substituted as defined herein with respect to the definitionof aryl. At least one —CH₂— group within any such cycloalkyl ring systemmay be optionally replaced with —C(O)—, —C(S)—, —C(═N—H)—, —C(═N—OH)—,—C(═N-alkyl)- (optionally substituted as defined herein with respect tothe definition of alkyl), or —C(═N—O-alkyl)- (optionally substituted asdefined herein with respect to the definition of alkyl).

Further examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, octahydronaphthyl, 2,3-dihydro-1H-indenyl, andthe like.

In an embodiment, a cycloalkyl may be selected from cyclopentyl,cyclohexyl, cycloheptyl, adamantenyl, bicyclo[2.2.1]heptyl, and thelike.

The cycloalkyl groups herein are unsubstituted or substituted in atleast one position with various groups. For example, such cycloalkylgroups may be optionally substituted with alkyl, alkoxy, —C(O)H,carboxy, alkoxycarbonyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,amido, alkanoylamino, amidino, alkoxycarbonylamino, N-alkyl amidino,N-alkyl amido, N,N′-dialkylamido, aralkoxycarbonylamino, halogen,alkylthio, alkylsulfinyl, alkylsulfonyl, hydroxy, cyano, nitro, amino,monoalkylamino, dialkylamino, haloalkyl, haloalkoxy, aminoalkyl,monoalkylaminoalkyl, dialkylaminoalkyl, and the like.

The term “cycloalkylcarbonyl” refers to an acyl group of the formulacycloalkyl-C(O)— in which the term “cycloalkyl” has the significancegiven above, such as cyclopropylcarbonyl, cyclohexylcarbonyl,adamantylcarbonyl, 1,2,3,4-tetrahydro-2-naphthoyl,2-acetamido-1,2,3,4-tetrahydro-2-naphthoyl,1-hydroxy-1,2,3,4-tetrahydro-6-naphthoyl, and the like.

The term “heterocycloalkyl,” “heterocycle,” or “heterocyclyl,” refers toa monocyclic, bicyclic or tricyclic heterocycle group, containing atleast one nitrogen, oxygen or sulfur atom ring member and having 3 to 8ring members in each ring, wherein at least one ring in theheterocycloalkyl ring system may optionally contain at least one doublebond. At least one —CH₂— group within any such heterocycloalkyl ringsystem may be optionally replaced with —C(O)—, —C(S)—, —C(N)—,—C(═N—H)—, —C(═N—OH)—, —C(═N-alkyl)- (optionally substituted as definedherein with respect to the definition of alkyl), or —C(═N—O-alkyl)-(optionally substituted as defined herein with respect to the definitionof alkyl).

The terms “bicyclic” and “tricyclic” as used herein are intended toinclude both fused ring systems, such as 2,3-dihydro-1H-indole, andsubstituted ring systems, such as bicyclohexyl. At least one —CH₂— groupwithin any such heterocycloalkyl ring system may be optionally replacedwith —C(O)—, —C(N)— or —C(S)—. Heterocycloalkyl is intended to includesulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, andcarbocyclic fused and benzo fused ring systems wherein the benzo fusedring system is optionally substituted as defined herein with respect tothe definition of aryl. Such heterocycloalkyl groups may be optionallysubstituted on one or more carbon atoms by halogen, alkyl, alkoxy,cyano, nitro, amino, alkylamino, dialkylamino, monoalkylaminoalkyl,dialkylaminoalkyl, haloalkyl, haloalkoxy, aminohydroxy, oxo, aryl,aralkyl, heteroaryl, heteroaralkyl, amidino, N-alkylamidino,alkoxycarbonylamino, alkylsulfonylamino, and the like, and/or on asecondary nitrogen atom (i.e., —NH—) by hydroxy, alkyl,aralkoxycarbonyl, alkanoyl, heteroaralkyl, phenyl, phenylalkyl, and thelike.

Examples of a heterocycloalkyl include morpholinyl, thiomorpholinyl,thiomorpholinyl S-oxide, thiomorpholinyl S,S-dioxide, piperazinyl,homopiperazinyl, pyrrolidinyl, pyrrolinyl, 2,5-dihydro-pyrrolyl,tetrahydropyranyl, pyranyl, thiopyranyl, piperidinyl, tetrahydrofuranyl,tetrahydrothienyl, imidazolidinyl, homopiperidinyl,1,2-dihydro-pyridinyl, homomorpholinyl, homothiomorpholinyl,homothiomorpholinyl S,S-dioxide, oxazolidinonyl, dihydropyrazolyl,dihydropyrrolyl, 1,4-dioxa-spiro[4.5]decyl, dihydropyrazinyl,dihydropyridinyl, dihydropyrimidinyl, dihydrofuryl, dihydropyranyl,tetrahydrothienyl S-oxide, tetrahydrothienyl S,S-dioxide,homothiomorpholinyl S-oxide, 2-oxo-piperidinyl, 5-oxo-pyrrolidinyl,2-oxo-1,2-dihydro-pyridinyl, 6-oxo-6H-pyranyl,1,1-dioxo-hexahydro-thiopyranyl, 1-acetyl-piperidinyl,1-methanesulfonylpiperidinyl, 1-ethanesulfonylpiperidinyl,1-oxo-hexahydro-thiopyranyl, 1-(2,2,2-trifluoroacetyl)-piperidinyl,1-formyl-piperidinyl, and the like.

In an embodiment, a heterocycloalkyl may be selected from pyrrolidinyl,2,5-dihydro-pyrrolyl, piperidinyl, 1,2-dihydro-pyridinyl, pyranyl,piperazinyl, imidazolidinyl, thiopyranyl, tetrahydropyranyl,1,4-dioxa-spiro[4.5]decyl, and the like.

In another embodiment, a heterocycloalkyl may be selected from2-oxo-piperidinyl, 5-oxo-pyrrolidinyl, 2-oxo-1,2-dihydro-pyridinyl,6-oxo-6H-pyranyl, 1,1-dioxo-hexahydro-thiopyranyl, 1-acetyl-piperidinyl,1-methanesulfonyl piperidinyl, 1-ethanesulfonylpiperidinyl,1-oxo-hexahydro-thiopyranyl, 1-(2,2,2-trifluoroacetyl)-piperidinyl;1-formyl-piperidinyl, and the like.

The term “aryl” refers to an aromatic carbocyclic group having a singlering (e.g., phenyl) or multiple condensed rings in which at least onering is aromatic. The aryl may be monocyclic, bicyclic, tricyclic, etc.Bicyclic and tricyclic as used herein are intended to include both fusedring systems, such as naphthyl and β-carbolinyl, and substituted ringsystems, such as biphenyl, phenylpyridyl, diphenylpiperazinyl,tetrahydronaphthyl, and the like. Preferred aryl groups of the presentinvention are phenyl, 1-naphthyl, 2-naphthyl, indanyl, indenyl,dihydronaphthyl, fluorenyl, tetralinyl or6,7,8,9-tetrahydro-5H-benzo[a]cycloheptenyl. The aryl groups herein areunsubstituted or substituted in one or more positions with variousgroups. For example, such aryl groups may be optionally substituted withalkyl, alkoxy, C(O)H, carboxy, alkoxycarbonyl, aryl, heteroaryl,cycloalkyl, heterocycloalkyl, amido, alkanoylamino, amidino,alkoxycarbonylamino, N-alkyl amidino, N-alkyl amido, N,N′-dialkylamido,aralkoxycarbonylamino, halogen, alkyl thio, alkylsulfinyl,alkylsulfonyl, hydroxy, cyano, nitro, amino, monoalkylamino,dialkylamino, aralkoxycarbonylamino, haloalkyl, haloalkoxy, aminoalkyl,monoalkylaminoalkyl, dialkylaminoalkyl, and the like.

Examples of aryl groups are phenyl, p-tolyl, 4-methoxyphenyl,4-(tert-butoxy)phenyl, 3-methyl-4-methoxyphenyl, 4-CF₃-phenyl,4-fluorophenyl, 4-chlorophenyl, 3-nitrophenyl, 3-aminophenyl,3-acetamidophenyl, 4-acetamidophenyl, 2-methyl-3-acetamidophenyl,2-methyl-3-aminophenyl, 3-methyl-4-aminophenyl, 2-amino-3-methylphenyl,2,4-dimethyl-3-aminophenyl, 4-hydroxyphenyl, 3-methyl-4-hydroxyphenyl,1-naphthyl, 2-naphthyl, 3-amino-1-naphthyl, 2-methyl-3-amino-1-naphthyl,6-amino-2-naphthyl, 4,6-dimethoxy-2-naphthyl, piperazinylphenyl, and thelike.

Further examples of aryl groups include 3-tert-butyl-1-fluoro-phenyl,1,3-difluoro-phenyl, (1-hydroxy-1-methyl-ethyl)-phenyl,1-fluoro-3-(2-hydroxy-1,1-dimethyl-ethyl)-phenyl,(1,1-dimethyl-propyl)-phenyl, cyclobutyl-phenyl, pyrrolidin-2-yl-phenyl,(5-oxo-pyrrolidin-2-yl)-phenyl, (2,5-dihydro-1H-pyrrol-2-yl)-phenyl,(1H-pyrrol-2-yl)-phenyl, (cyano-dimethyl-methyl)-phenyl,tert-butyl-phenyl, 1-fluoro-2-hydroxy-phenyl,1,3-difluoro-4-propylamino-phenyl, 1,3-difluoro-4-hydroxy-phenyl,1,3-difluoro-4-ethylamino-phenyl, 3-isopropyl-phenyl,(3H-[1,2,3]triazol-4-yl)-phenyl, [1,2,3]triazol-1-yl-phenyl,[1,2,4]thiadiazol-3-yl-phenyl, [1,2,4]thiadiazol-5-yl-phenyl,(4H-[1,2,4]triazol-3-yl)-phenyl, [1,2,4]oxadiazol-3-yl-phenyl,imidazol-1-yl-phenyl, (3H-imidazol-4-yl)-phenyl,[1,2,4]triazol-4-yl-phenyl, [1,2,4]oxadiazol-5-yl-phenyl,isoxazol-3-yl-phenyl, (1-methyl-cyclopropyl)-phenyl,isoxazol-4-yl-phenyl, isoxazol-5-yl-phenyl, 1-cyano-2-tert-butyl-phenyl,1-trifluoromethyl-2-tert-butyl-phenyl, 1-chloro-2-tert-butyl-phenyl,1-acetyl-2-tert-butyl-phenyl, 1-tert-butyl-2-methyl-phenyl,1-tert-butyl-2-ethyl-phenyl, 1-cyano-3-tert-butyl-phenyl,1-trifluoromethyl-3-tert-butyl-phenyl, 1-chloro-3-tert-butyl-phenyl,1-acetyl-3-tert-butyl-phenyl, 1-tert-butyl-3-methyl-phenyl1-tert-butyl-3-ethyl-phenyl, 4-tert-butyl-1-imidazol-1-yl-phenyl,ethylphenyl, isobutylphenyl, isopropylphenyl,3-allyloxy-1-fluoro-phenyl, (2,2-dimethyl-propyl)-phenyl, ethynylphenyl,1-fluoro-3-heptyloxy-phenyl,1-fluoro-3-[2-(2-methoxy-ethoxy)-ethoxy]-phenyl,1-benzyloxy-3-fluoro-phenyl, 1-fluoro-3-hydroxy-phenyl,1-fluoro-3-hexyloxy-phenyl, (4-methyl-thiophen-2-yl)-phenyl,(5-acetyl-thiophen-2-yl)-phenyl, furan-3-yl-phenyl,thiophen-3-yl-phenyl, (5-formyl-thiophen-2-yl)-phenyl,(3-formyl-furan-2-yl)-phenyl, acetylamino-phenyl, trifluoromethylphenyl,sec-butyl-phenyl, pentylphenyl, (3-methyl-butyl)-phenyl,(1-ethyl-propyl)-phenyl, cyclopentyl-phenyl, 3-pent-4-enyl-phenyl,phenyl propionic acid ethyl ester, pyridin-2-yl-phenyl,(3-methyl-pyridin-2-yl)-phenyl, thiazol-2-yl-phenyl,(3-methyl-thiophen-2-yl)-phenyl, fluoro-phenyl, adamantan-2-yl-phenyl,1,3-difluoro-2-hydroxy-phenyl, cyclopropyl-phenyl,1-bromo-3-tert-butyl-phenyl, (3-bromo-[1,2,4]thiadiazol-5-yl)-phenyl,(1-methyl-1H-imidazol-2-yl)-phenyl,3,5-dimethyl-3H-pyrazol-4-yl)-phenyl,(3,6-dimethyl-pyrazin-2-yl)-phenyl, (3-cyano-pyrazin-2-yl)-phenyl,thiazol-4-yl-phenyl, (4-cyano-pyridin-2-yl)-phenyl, pyrazin-2-yl-phenyl,(6-methyl-pyridazin-3-yl)-phenyl, (2-cyano-thiophen-3-yl)-phenyl,(2-chloro-thiophen-3-yl)-phenyl, (5-acetyl-thiophen-3-yl)-phenyl,cyano-phenyl, and the like.

The term “heteroaryl” refers to an aromatic heterocycloalkyl group asdefined above. The heteroaryl groups herein are unsubstituted orsubstituted in at least one position with various groups. For example,such heteroaryl groups may be optionally substituted with, for example,alkyl, alkoxy, halogen, hydroxy, cyano, nitro, amino, monoalkylamino,dialkylamino, haloalkyl, haloalkoxy, C(O)H, carboxy, alkoxycarbonyl,cycloalkyl, heterocycloalkyl, aryl, heteroaryl, amido, alkanoylamino,amidino, alkoxycarbonylamino, N-alkyl amidino, N-alkyl amido,N,N′-dialkylamido, alkyl thio, alkylsulfinyl, alkylsulfonyl,aralkoxycarbonylamino, aminoalkyl, monoalkylaminoalkyl,dialkylaminoalkyl, and the like.

Examples of heteroaryl groups include pyridyl, pyrimidyl, furanyl,imidazolyl, thienyl, oxazolyl, thiazolyl, pyrazinyl, 3-methyl-thienyl,4-methyl-thienyl, 3-propyl-thienyl, 2-chloro-thienyl,2-chloro-4-ethyl-thienyl, 2-cyano-thienyl, 5-acetyl-thienyl,5-formyl-thienyl, 3-formyl-furanyl, 3-methyl-pyridinyl,3-bromo-[1,2,4]thiadiazolyl, 1-methyl-1H-imidazole,3,5-dimethyl-3H-pyrazolyl, 3,6-dimethyl-pyrazinyl, 3-cyano-pyrazinyl,4-tert-butyl-pyridinyl, 4-cyano-pyridinyl, 6-methyl-pyridazinyl,2-tert-butyl-pyrimidinyl, 4-tert-butyl-pyrimidinyl,6-tert-butyl-pyrimidinyl, 5-tert-butyl-pyridazinyl,6-tert-butyl-pyridazinyl, quinolinyl, benzothienyl, indolyl, indolinyl,pyridazinyl, isoindolyl, isoquinolyl, quinazolinyl, quinoxalinyl,phthalazinyl, imidazolyl, isoxazolyl, pyrazolyl, indolizinyl, indazolyl,benzothiazolyl, benzimidazolyl, benzofuranyl, thienyl, pyrrolyl,oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, oxazolopyridinyl,imidazopyridinyl, isothiazolyl, naphthyridinyl, cinnolinyl, carbazolyl,beta-carbolinyl, isochromanyl, chromanyl, tetrahydroisoquinolinyl,isoindolinyl, isobenzotetrahydrofuranyl, isobenzotetrahydrothienyl,isobenzothienyl, benzoxazolyl, pyridopyridinyl, benzotetrahydrofuranyl,benzotetrahydrothienyl, purinyl, benzodioxolyl, triazinyl, phenoxazinyl,phenothiazinyl, pteridinyl, benzothiazolyl, imidazopyridinyl,imidazothiazolyl, dihydrobenzisoxazinyl, benzisoxazinyl, benzoxazinyl,dihydrobenzisothiazinyl, benzopyranyl, benzothiopyranyl, coumarinyl,isocoumarinyl, chromonyl, chromanonyl, pyridinyl-N-oxide,tetrahydroquinolinyl, dihydroquinolinyl, dihydroquinolinonyl,dihydroisoquinolinonyl, dihydrocoumarinyl, dihydroisocoumarinyl,isoindolinonyl, benzodioxanyl, benzoxazolinonyl, pyrrolyl N-oxide,pyrimidinyl N-oxide, pyridazinyl N-oxide, pyrazinyl N-oxide, quinolinylN-oxide, indolyl N-oxide, indolinyl N-oxide, isoquinolyl N-oxide,quinazolinyl N-oxide, quinoxalinyl N-oxide, phthalazinyl N-oxide,imidazolyl N-oxide, isoxazolyl N-oxide, oxazolyl N-oxide, thiazolylN-oxide, indolizinyl N-oxide, indazolyl N-oxide, benzothiazolyl N-oxide,benzimidazolyl N-oxide, pyrrolyl N-oxide, oxadiazolyl N-oxide,thiadiazolyl N-oxide, triazolyl N-oxide, tetrazolyl N-oxide,benzothiopyranyl S-oxide, benzothiopyranyl S,S-dioxide,tetrahydrocarbazole, tetrahydrobetacarboline, and the like.

In an embodiment, a heteroaryl group may be selected from pyridyl,pyrimidyl, furanyl, imidazolyl, thienyl, oxazolyl, thiazolyl, pyrazinyl,and the like.

In another embodiment, a heteroaryl group may be selected from3-methyl-thienyl, 4-methyl-thienyl, 3-propyl-thienyl, 2-chloro-thienyl,2-chloro-4-ethyl-thienyl, 2-cyano-thienyl, 5-acetyl-thienyl,5-formyl-thienyl, 3-formyl-furanyl, 3-methyl-pyridinyl,3-bromo-[1,2,4]thiadiazolyl, 1-methyl-1H-imidazole,3,5-dimethyl-3H-pyrazolyl, 3,6-dimethyl-pyrazinyl, 3-cyano-pyrazinyl,4-tert-butyl-pyridinyl, 4-cyano-pyridinyl, 6-methyl-pyridazinyl,2-tert-butyl-pyrimidinyl, 4-tert-butyl-pyrimidinyl,6-tert-butyl-pyrimidinyl, 5-tert-butyl-pyridazinyl,6-tert-butyl-pyridazinyl, and the like.

Further examples of heterocycloalkyls and heteroaryls may be found inKatritzky, A. R. et al., Comprehensive Heterocyclic Chemistry: TheStructure, Reactions, Synthesis and Use of Heterocyclic Compounds, Vol.1-8, New York: Pergamon Press, 1984.

The term “aralkoxycarbonyl” refers to a group of the formulaaralkyl-O—C(O)— in which the term “aralkyl” is encompassed by thedefinitions above for aryl and alkyl. Examples of an aralkoxycarbonylgroup include benzyloxycarbonyl 4-methoxyphenylmethoxycarbonyl, and thelike.

The term “aryloxy” refers to a group of the formula —O-aryl in which theterm aryl is as defined above.

The term “aralkanoyl” refers to an acyl group derived from anaryl-substituted alkanecarboxylic acid such as phenylacetyl,3-phenylpropionyl(hydrocinnamoyl), 4-phenylbutyryl, (2-naphthyl)acetyl,4-chlorohydrocinnamoyl, 4-aminohydrocinnamoyl, 4-methoxyhydrocinnamoyl,and the like.

The term “aroyl” refers to an acyl group derived from an arylcarboxylicacid, “aryl” having the meaning given above. Examples of such aroylgroups include substituted and unsubstituted benzoyl or naphthoyl suchas benzoyl, 4-chlorobenzoyl, 4-carboxybenzoyl,4-(benzyloxycarbonyl)benzoyl, 1-naphthoyl, 2-naphthoyl, 6-carboxy-2naphthoyl, 6-(benzyloxycarbonyl)-2-naphthoyl, 3-benzyloxy-2-naphthoyl,3-hydroxy-2-naphthoyl, 3-(benzyloxyformamido)-2-naphthoyl, and the like.

The term “haloalkyl” refers to an alkyl group having the meaning asdefined above wherein one or more hydrogens are replaced with a halogen.Examples of such haloalkyl groups include chloromethyl, 1-bromoethyl,fluoromethyl, difluoromethyl, trifluoromethyl, 1,1,1-trifluoroethyl, andthe like.

The term “epoxide” refers to chemical compounds or reagents comprising abridging oxygen wherein the bridged atoms are also bonded to one anothereither directly or indirectly. Examples of epoxides include epoxyalkyl(e.g., ethylene oxide, and 1,2-epoxybutane), and epoxycycloalkyl (e.g.,1,2-epoxycyclohexane, 1,2-epoxy-1-methylcyclohexane), and the like.

The term “structural characteristics” refers to chemical moieties,chemical motifs, and portions of chemical compounds. These include Rgroups, such as but not limited to those defined herein, ligands,appendages, and the like. For example, structural characteristics may bedefined by their properties, such as, but not limited to, their abilityto participate in intermolecular interactions including Van der Waal'sinteractions (e.g., electrostatic interactions, dipole-dipoleinteractions, dispersion forces, hydrogen bonding, and the like). Suchcharacteristics may impart desired pharmacokinetic properties and thushave an increased ability to cause the desired effect and thus preventor treat the targeted diseases or conditions.

Compounds of formula (I) also comprise structural moieties that mayparticipate in inhibitory interactions with at least one subsite ofbeta-secretase. For example, moieties of the compounds of formula (I)may interact with at least one of the S1, S1′ and S2′ subsites, whereinS1 comprises residues Leu30, Tyr71, Phe108, Ile110, and Trp115, S1′comprises residues Tyr198, Ile226, Val227, Ser 229, and Thr231, and S2′comprises residues Ser35, Asn37, Pro70, Tyr71, Ile118, and Arg128. Suchcompounds and methods of treatment may have an increased ability tocause the desired effect and thus prevent or treat the targeted diseasesor conditions.

The term “pharmaceutically acceptable” refers to those properties and/orsubstances that are acceptable to the patient from apharmacological/toxicological point of view, and to the manufacturingpharmaceutical chemist from a physical/chemical point of view regardingcomposition, formulation, stability, patient acceptance, andbioavailability.

The term “effective amount” as used herein refers to an amount of atherapeutic agent administered to a host, as defined herein, necessaryto achieve a desired effect.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent administered to a host to treat or preventa condition treatable by administration of a composition of theinvention. That amount is the amount sufficient to reduce or lessen atleast one symptom of the disease being treated or to reduce or delayonset of one or more clinical markers or symptoms of the disease.

The term “therapeutically active agent” refers to a compound orcomposition that is administered to a host, either alone or incombination with another therapeutically active agent, to treat orprevent a condition treatable by administration of a composition of theinvention.

The terms “pharmaceutically acceptable salt” and “salts thereof” referto acid addition salts or base addition salts of the compounds in thepresent invention. A pharmaceutically acceptable salt is any salt whichretains the activity of the parent compound and does not impart anydeleterious or undesirable effect on the subject to whom it isadministered and in the context in which it is administered.Pharmaceutically acceptable salts include salts of both inorganic andorganic acids. Pharmaceutically acceptable salts include acid salts suchas acetic, aspartic, benzenesulfonic, benzoic, bicarbonic, bisulfuric,bitartaric, butyric, calcium edetate, camsylic, carbonic, chlorobenzoic,citric, edetic, edisylic, estolic, esyl, esylic, formic, fumaric,gluceptic, gluconic, glutamic, glycolylarsanilic, hexamic,hexylresorcinoic, hydrabamic, hydrobromic, hydrochloric, hydroiodic,hydroxynaphthoic, isethionic, lactic, lactobionic, maleic, malic,malonic, mandelic, methanesulfonic, methylnitric, methylsulfuric, mucic,muconic, napsylic, nitric, oxalic, p-nitromethanesulfonic, pamoic,pantothenic, phosphoric, monohydrogen phosphoric, dihydrogen phosphoric,phthalic, polygalactouronic, propionic, salicylic, stearic, succinic,sulfamic, sulfanilic, sulfonic, sulfuric, tannic, tartaric, teoclic,toluenesulfonic, and the like. Other acceptable salts may be found, forexample, in Stahl et al., Pharmaceutical Salts: Properties, Selection,and Use, Wiley-VCH; 1st edition (Jun. 15, 2002).

In an embodiment of the present invention, a pharmaceutically acceptablesalt is selected from hydrochloric, hydrobromic, hydroiodic, nitric,sulfuric, phosphoric, citric, methanesulfonic, CH₃—(CH₂)₀₋₄—COOH,HOOC—(CH₂)₀₋₄—COOH, HOOC—CH═CH—COOH, phenyl-COOH, and the like.

The term “unit dosage form” refers to physically discrete units suitableas unitary dosages for human subjects or other mammals, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect, in association with a suitablepharmaceutical vehicle. The concentration of active compound in the drugcomposition will depend on absorption, inactivation, and/or excretionrates of the active compound, the dosage schedule, the amountadministered and medium and method of administration, as well as otherfactors known to those of skill in the art.

The term “modulate” refers to a chemical compound's activity of eitherenhancing or inhibiting a functional property of biological activity orprocess.

The terms “interact” and “interactions” refer to a chemical compound'sassociation and/or reaction with another chemical compound, such as aninteraction between an inhibitor and beta-secretase. Interactionsinclude, but are not limited to, hydrophobic, hydrophilic, lipophilic,lipophobic, electrostatic, and van der Waal's interactions includinghydrogen bonding.

An “article of manufacture” as used herein refers to materials usefulfor the diagnosis, prevention or treatment of the disorders describedabove, such as a container with a label. The label can be associatedwith the article of manufacture in a variety of ways including, forexample, the label may be on the container or the label may be in thecontainer as a package insert. Suitable containers include, for example,blister packs, bottles, bags, vials, syringes, test tubes, and the like.The containers may be formed from a variety of materials such as glass,metal, plastic, rubber, paper, and the like. The container holds acomposition as described herein which is effective for diagnosing,preventing, or treating a condition treatable by a compound orcomposition of the present invention.

The article of manufacture may contain bulk quantities or less of acomposition as described herein. The label on, or associated with, thecontainer may provide instructions for the use of the composition indiagnosing, preventing, or treating the condition of choice,instructions for the dosage amount and for the methods ofadministration. The label may further indicate that the composition isto be used in combination with one or more therapeutically active agentswherein the therapeutically active agent is selected from anantioxidant, an anti-inflammatory, a gamma-secretase inhibitor, aneurotrophic agent, an acetyl cholinesterase inhibitor, a statin, anA-beta, an anti-A-beta antibody, and/or a beta-secretase complex orfragment thereof. The article of manufacture may further comprisemultiple containers, also referred to herein as a kit, comprising atherapeutically active agent or a pharmaceutically-acceptable buffer,such as phosphate-buffered saline, Ringer's solution and/or dextrosesolution. It may further include other materials desirable from acommercial and user standpoint, including other buffers, diluents,filters, needles, syringes, and/or package inserts with instructions foruse.

The compounds of formula (I), their compositions, and methods oftreatment employing them, can be enclosed in multiple or single dosecontainers. The enclosed compounds and/or compositions can be providedin kits, optionally including component parts that can be assembled foruse. For example, a compound inhibitor in lyophilized form and asuitable diluent may be provided as separated components for combinationprior to use. A kit may include a compound inhibitor and at least oneadditional therapeutic agent for co-administration. The inhibitor andadditional therapeutic agents may be provided as separate componentparts.

A kit may include a plurality of containers, each container holding atleast one unit dose of the compound of the present invention. Thecontainers are preferably adapted for the desired mode ofadministration, including, for example, pill, tablet, capsule, powder,gel or gel capsule, sustained-release capsule, or elixir form, and/orcombinations thereof, and the like for oral administration, depotproducts, pre-filled syringes, ampoules, vials, and the like forparenteral administration, and patches, medipads, creams, and the likefor topical administration.

The term “C_(max)” refers to the peak plasma concentration of a compoundin a host.

The term “T_(max)” refers to the time at peak plasma concentration of acompound in a host.

The term “half-life” refers to the period of time required for theconcentration or amount of a compound in a host to be reduced to exactlyone-half of a given concentration or amount.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

The present invention is directed to novel compounds and also to methodsof treating at least one condition, disorder, or disease associated withamyloidosis using such compounds. Amyloidosis refers to a collection ofdiseases, disorders, or conditions associated with abnormal depositionof amyloidal protein.

An embodiment of the present invention is to provide methods ofpreventing or treating at least one condition associated withamyloidosis using compounds of formula (I) with a high degree ofefficacy. Compounds and methods of treatment that are efficacious arethose that have an increased ability to cause the desired effect andthus prevent or treat the targeted diseases or conditions.

Another embodiment of the present invention is to provide compounds offormula (I),

or pharmaceutically acceptable salts thereof, for preventing or treatingat least one condition that benefits from inhibition of at least oneaspartyl-protease, and wherein R₁, R₂, and R_(C) are defined below.

Another embodiment of the present invention is to provide methods forpreventing or treating at least one condition that benefits frominhibition of at least one aspartyl-protease, comprising compounds offormula (I), or pharmaceutically acceptable salts thereof, wherein theinhibition is at least 10% for a dose of 100 mg/kg or less, and whereinR₁, R₂, and R_(C) are defined below.

Another embodiment of the present invention is to provide a method ofpreventing or treating at least one condition that benefits frominhibition of at least one aspartyl-protease, comprising administeringto a host a composition comprising a therapeutically effective amount ofat least one compound of formula (I), or pharmaceutically acceptablesalts thereof, wherein

-   R₁ is

-   -   wherein n is 0 or 1; q is 0 or 1; r is 0, 1, or 2;    -   K is selected from —(CR_(3a)R_(3b))—, —O—, —SO₂—, —C(O)—, and        —CH(NR₅₅R₆₀)—;    -   R₅₅ and R₆₀ are each independently selected from hydrogen and        alkyl;    -   R_(3a) and R_(3b) are independently selected from hydrogen,        halogen, —O-alkyl, and alkyl optionally substituted with at        least one group independently selected from halogen, —CN, —CF₃,        and —OH;    -   W is selected from —(CH₂)₁₋₄—, —O—, —S(O)₀₋₂—, —N(R₅₅)—, and        —C(O)—;    -   E is a bond or alkyl;    -   A is selected from aryl optionally substituted with at least one        group independently selected from R₅₀, cycloalkyl optionally        substituted with at least one group independently selected from        R₅₀, heteroaryl optionally substituted with at least one group        independently selected from R₅₀, and heterocycle optionally        substituted with at least one group independently selected from        R₅₀,        -   wherein at least one atom of the heterocycle is optionally            replaced with —C(O)— and —S(O)₀₋₂—,        -   wherein at least one heteroatom of the heteroaryl or            heterocycle is optionally substituted with a group            independently selected from —(CO)₀₋₁R₂₁₅, —(CO)₀₋₁R₂₂₀,            —S(O)₀₋₂R₂₀₀, and —N(R₂₀₀)—S(O)₀₋₂R₂₀₀;    -   R₅₀ is independently selected from —OH, halogen, —OCF₃, —NO₂,        —CN, —N(R)C(O)R, —CO₂—R, —NH—CO₂—R, —O-(alkyl)-CO₂H, —NRR′, —SR,        —CH₂OH, —C(O)—R₂₅, —C(O)NRR′, —SO₂NRR′, —S(O)₁₋₂R₂₅, alkyl        (optionally substituted with at least one group independently        selected from —CF₃, halogen, —O-alkyl, —OCF₃, —NRR′, —OH, and        —CN), cycloalkyl (optionally substituted with at least one group        independently selected from —CF₃, halogen, —O-alkyl, —OCF₃,        —NRR′, —OH, and —CN), —O-alkyl (optionally substituted with at        least one group independently selected from —CF₃, halogen,        —O-alkyl, —OCF₃, —NRR′, —OH, and —CN), —O-benzyl (optionally        substituted with at least one group independently selected from        —H, —OH, halogen, and alkyl), —O—(CH₂)₀₋₂—O—(CH₂)₁₋₂—O-alkyl,        and —(CH₂)₀₋₂—O—(CH₂)₁₋₂—OH;    -   R and R′ are each independently selected from hydrogen, alkyl,        —(CH₂)₀₋₂-aryl and —(CH₂)₀₋₂-cycloalkyl, wherein each aryl or        cycloalkyl is optionally substituted with at least one group        independently selected from halogen, hydroxy, alkyl, —O-alkyl,        amino, monoalkylamino, and dialkylamino;    -   R₂₅ is selected from alkyl, —(CH₂)₀₋₂-aryl and        —(CH₂)₀₋₂-cycloalkyl, wherein each aryl or cycloalkyl is        optionally substituted with at least one group independently        selected from halogen, hydroxy, alkyl, —O-alkyl, amino,        monoalkylamino, and dialkylamino;    -   L is selected from a bond, —C(O)—, —S(O)₁₋₂—, —O—,        —C(R₁₁₀)(R₁₁₂)O—, —OC(R₁₁₀)(R₁₁₂)—, —N(R₁₁₀)—, —C(O)N(R₁₁₀)—,        —N(R₁₁₀)C(O)—, —C(R₁₁₀)(R′)—, —C(OH)R₁₁₀—, —SO₂NR₁₁₀—,        —N(R₁₁₀)SO₂—, —N(R₁₁₀)C(O)N(R₁₁₂)—, —N(R₁₁₀)C(S)N(R₁₁₂)—,        —OCO₂—, —NCO₂—, and —OC(O)N(R₁₁₀)—;    -   R₁₁₀ and R₁₁₂ are each independently selected from hydrogen and        alkyl optionally substituted with at least one group        independently selected from —OH, —O-alkyl, and halogen;    -   G is selected from        -   -alkyl optionally substituted with at least one group            independently selected from —CO₂H, —CO₂(alkyl), —O-alkyl,            —OH, —NRR′, alkyl, haloalkyl, -alkyl-O-alkyl, aryl            (optionally substituted with at least one group            independently selected from R₅₀), and heteroaryl (optionally            substituted with at least one group independently selected            from R₅₀);        -   —(CH₂)₀₋₃-cycloalkyl wherein cycloalkyl is optionally            substituted with at least one group independently selected            from —CO₂H, —CO₂— (alkyl), —O-alkyl, —OH, —NH₂, haloalkyl,            alkyl, -alkyl-O-alkyl, mono(alkyl)amino, di(alkyl)amino,            aryl (optionally substituted with at least one group            independently selected from R₅₀), and heteroaryl (optionally            substituted with at least one group independently selected            from R₅₀);        -   —(CRR)₀₋₄-aryl wherein the aryl is optionally substituted            with at least one group independently selected from R₅₀;        -   —(CH₂ ₀₋₄-heteroaryl wherein the heteroaryl is optionally            substituted with at least one group independently selected            from R₅₀;        -   —(CH₂)₀₋₄-heterocycle, wherein the heterocycle is optionally            substituted with at least one group independently selected            from R₅₀; and        -   —C(R₁₀)(R₁₂)—C(O)—NH—R₁₄;        -   R₁₀ and R₁₂ are each independently selected from —H, alkyl,            -(alkyl)₀₋₁-aryl, -(alkyl)₀₋₁-heteroaryl,            -(alkyl)₀₋₁-heterocycle, aryl, heteroaryl, heterocycle,            —(CH₂)₁₋₄—OH, —(CH₂)₁₋₄-Z-(CH₂)₁₋₄-aryl, and            —(CH₂)₁₋₄-Z-(CH₂)₁₋₄-heteroaryl; wherein the heterocycle,            aryl, and heteroaryl groups included within R₁₀ and R₁₂ are            optionally substituted with at least one group independently            selected from R₅₀;        -   Z is selected from —O—, —S—, and —NR₁₆—;            -   R₁₄ is selected from —H, alkyl, aryl, heteroaryl,                heterocycle, -(alkyl)-aryl, -(alkyl)-heteroaryl, alkyl,                and —(CH₂)₀₋₂—O—(CH₂)₀₋₂—OH; wherein the heterocycle,                aryl, and heteroaryl groups included within R₁₄ are                optionally substituted with at least one group                independently selected from R₅₀,        -   R₁₆ is selected from hydrogen and alkyl; or

-   R₁ is selected from

-   -   alkyl;    -   wherein    -   X, Y, and Z are independently selected from —C(H)₀₋₂—, —O—,        —C(O)—, —NH—, and —N—;        -   wherein at least one bond of the (IIf) ring may optionally            be a double bond;    -   R₅₀, R_(50a), and R_(50b) are independently selected from —H,        halogen, —OH, —SH, —CN, —C(O)-alkyl, —NR₇R₈, —NO₂,        —S(O)₀₋₂-alkyl, alkyl, alkoxy, —O-benzyl (optionally substituted        with at least one group independently selected from —H, —OH, and        alkyl), —C(O)—NR₇R₈, alkyloxy, alkoxyalkoxyalkoxy, and        cycloalkyl;        -   wherein the alkyl, alkoxy, and cycloalkyl groups within R₅₀,            R_(50a), and R_(50b) are optionally substituted with at            least one group independently selected from alkyl, halogen,            OH, NR₅R₆, CN, haloalkoxy, NR₇R₈, and alkoxy;    -   R₅ and R₆ are independently selected from —H and alkyl, or    -   R₅ and R₆, and the nitrogen to which they are attached, form a 5        or 6 membered heterocycloalkyl ring; and

-   R₇ and R₈ are independently selected from —H, alkyl optionally    substituted with at least one group independently selected from —OH,    —NH₂, and halogen, -cycloalkyl, and -alkyl-O-alkyl;

-   R₂ is selected from H, —OH, —O-alkyl (optionally substituted with at    least one group independently selected from R₂₀₀), —O-aryl    (optionally substituted with at least one group independently    selected from R₂₀₀), alkyl (optionally substituted with at least one    group independently selected from R₂₀₀), —NH-alkyl (optionally    substituted with at least one group independently selected from    R₂₀₀), heterocycloalkyl, (wherein at least one carbon is optionally    replaced with a group independently selected from —(CR₂₄₅R₂₅₀)—,    —O—, —C(O)—, —C(O)C(O)—, —N(R₂₀₀)₀₋₂—, and —S(O)₀₋₂—, and wherein    the heterocycloalkyl is optionally substituted with at least one    group independently selected from R₂₀₀), —NH-heterocycloalkyl,    (wherein at least one carbon is optionally replaced with a group    independently selected from —(CR₂₄₅R₂₅₀)—, —O—, —C(O)—, —C(O)C(O)—,    —N(R₂₀₀)₀₋₁—, and —S(O)₀₋₂—, and wherein the heterocycloalkyl is    optionally substituted with at least one group independently    selected from R₂₀₀), —C(O)—N(R₃₁₅)(R₃₂₀), (wherein R₃₁₅ and R₃₂₀ are    each independently selected from H, alkyl, and aryl),    —O—C(O)—N(R₃₁₅)(R₃₂₀), —NH—R₄₀₀, R₄₀₀, —NH—R₅₀₀, R₅₀₀, —NH—R₆₀₀,    R₆₀₀, and —NH—R₇₀₀;

-   R₄₀₀ is

-    wherein R₄₀₅ is selected from H, —N(R₅₁₅)₂ and O-alkyl;-   R₅₀₀ is a heteroaryl selected from III(a) and III(b)

-    wherein    -   M₁ and M₄ are independently selected from —C(R₅₀₅)—, —N—,        —N(R₅₁₅)—, —S—, and —O—;    -   M₂ and M₃ are independently selected from —C(R₅₁₀)—,        —N(R₅₂₀)₀₋₁—, —S—, and —O—;    -   M₅ is selected from —C— and —N—;        -   R₅₀₅ is independently selected from H, alkyl, halogen, —NO₂,            —CN, —R₂₀₀, and aryl;        -   R₅₁₀ is independently selected from H, alkyl, halogen,            amino, —CF₃, —R₂₀₀, and aryl;        -   R₅₁₅ is independently selected from H, alkyl, and aryl;        -   R₅₂₀ is independently selected from H, alkyl,            —(CH₂)₀₋₂-aryl, and —C(Ph)₃;-   R₆₀₀ is a monocyclic, bicyclic, or tricyclic heteroaryl ring system    of 6, 7, 8, 9, 10, 11, 12, 13, or 14 atoms, optionally substituted    with at least one group independently selected from R₆₀₅;    -   R₆₀₅ is selected from hydrogen, halogen, alkyl, aryl,        —CO₂-alkyl, nitro, —CN, amino, —NR₂₂₀R₂₂₅, -thioalkyl, —CF₃,        —OH, —O-alkyl, and heterocycloalkyl;-   R₇₀₀ is aryl optionally substituted with at least one R₂₀₅;-   R_(C) is selected from formula (IIIa), (IIIb), (IIIc), (IIId),    (IIIe), and (IIIf)

-    wherein,    -   A₁ and A₂ are independently selected from —(CH₂)₁₋₂—,        —CH(R₂₀₀)—, —C(R₂₀₀)₂—, —NH—, —NR₂₂₀—, —C(═N—R₂₃₀)—,        —C(═CH—R₂₃₀)—, —C(═N—C(O)—R₂₃₀)—, and —C(═CH—C(O)—R₂₃₀)—;    -   A₃, A₄, A₅, and A₆ are independently selected from —CH₂—,        —CH(R₂₀₀)—, —C(R₂₀₀)₂—, —O—, —C(O)—, —S(O)₀₋₂—, —NH—, —NR₂₂₀—,        —N(CO)₀₋₁R₂₀₀—, —N(S(O₂)alkyl)-, —C(═N—R₂₃₀)—,        —C(═N—NH(alkyl))-, —C(═N—N(alkyl)(alkyl))-,        —C(═N—O—(CH₂)₁₋₄—OH)—, —C(═CH—R₂₃₀)—, —C(═N—C(O)—R₂₃₀)—, and        —C(═CH—C(O)—R₂₃₀)—;    -   R₂₃₀ is independently selected from —H, —OH, R₂₁₅ (optionally        substituted with —OH, —NH₂, —C(O)H, and —CN), alkyl, cycloalkyl,        alkoxy, -alkyl-OH, -alkyl-NH₂, -alkyl-C(O)H, —O—R₂₁₅ (optionally        substituted with —OH, —NH₂, —C(O)H, and —CN), —O-alkyl,        —O-alkyl-OH, —O-alkyl-NH₂, —O-alkyl-C(O)H, —NH₂, —NHR₂₁₅,        —N(R₂₁₅)₂, —NR₂₃₅R₂₄₀, and —CN;        -   wherein at least one carbon of the alkyl or cycloalkyl            within R₂₃₀ is optionally independently replaced with —C(O)—            or a heteroatom;    -   wherein the cycloalkyl and heterocylcoalkyl within formulae        (IIIa), (IIIb), (IIIc), (IIId), (IIIe), and (IIIf) may        optionally contain at least one double bond;    -   wherein in formulae (IIIa), (IIIb), (IIIc), and (IIId), at least        one of A₁, A₂, A₃, A₄, or A₅ is selected from —C(═N—R₂₃₀)—,        —C(═N—NH(alkyl))-, —C(═N—N(alkyl)(alkyl))-,        C(═N—O—(CH₂)₁₋₄—OH)—, —C(═CH—R₂₃₀)—, —C(═N—C(O)—R₂₃₀)—, and        —C(═CH—C(O)—R₂₃₀)—;    -   wherein in formulae (IIIe) and (IIIf), when A₁, A₂, and A₆ are        selected from —(CH₂)₀₋₂—, —CH(R₂₀₀)—, —C(R₂₀₀)₂—, —O—, —C(O)—,        —S(O)₀₋₂—, —NH—, —NR₂₂₀—, —N(CO)₀₋₁R₂₀₀—, and —N(S(O₂)alkyl)-,        at least one carbon of the aryl ring group within (IIIe) and        (IIIf) is optionally independently replaced with a group        selected from —N—, —NH—, —O—, —C(O)—, and —S(O)₀₋₂—;    -   wherein each aryl or heteroaryl group attached directly or        indirectly to R_(C) is optionally substituted with at least one        group independently selected from R₂₀₀;    -   wherein each cycloalkyl or heterocycloalkyl attached directly or        indirectly to R_(C) is optionally substituted with at least one        group independently selected from R₂₁₀; and    -   R_(x) is selected from        -   -aryl, heteroaryl, cycloalkyl, heterocycloalkyl, and            —R_(xa)—R_(xb),        -   wherein R_(xa) and R_(xb) are independently selected from            -aryl, -heteroaryl, -cycloalkyl, and -heterocycloalkyl;        -   wherein each aryl or heteroaryl group within R_(X) is            optionally substituted with at least one group independently            selected from R₂₀₀;        -   wherein each cycloalkyl or heterocycloalkyl within R_(X) is            optionally substituted with at least one group independently            selected from R₂₁₀; and        -   wherein at least one carbon of the heteroaryl or            heterocycloalkyl group within R_(X) is independently            optionally replaced with a group independently selected from            —NH—, —N—, —N(CO)₀₋₁R₂₁₅—, —N(CO)₀₋₁R₂₂₀—, —O—, —C(O)—,            —S(O)₀₋₂—, and —NS(O)₀₋₂R₂₀₀;    -   R₂₀₀ at each occurrence is independently selected from        -   -alkyl optionally substituted with at least one group            independently selected from R₂₀₅, —OH, —NO₂, -halogen, —CN,            —(CH₂)₀₋₄—C(O)H, —(CO)₀₋₁R₂₁₅, —(CO)₀₋₁R₂₂₀,            —(CH₂)₀₋₄—(CO)₀₋₁—NR₂₂₀R₂₂₅, —(CH₂)₀₋₄—(CO)₀₋₁—NH(R₂₁₅),            —(CH₂)₀₋₄—C(O)-alkyl, —(CH₂)₀₋₄—(CO)₀₋₁-cycloalkyl,            —(CH₂)₀₋₄—(CO)₀₋₁-heterocycloalkyl, —(CH₂)₀₋₄—(CO)₀₋₁-aryl,            —(CH₂)₀₋₄—(CO)₀₋₁-heteroaryl, —(CH₂)₀₋₄—C(O)—O—R₂₁₅,            —(CH₂)₀₋₄—SO₂—NR₂₂₀R₂₂₅, —(CH₂)₀₋₄—S(O)₀₋₂-alkyl,            —(CH₂)₀₋₄—S(O)₀₋₂-cycloalkyl, —(CH₂)₀₋₄—N(H or            R₂₁₅)—C(O)—O—R₂₁₅, —(CH₂)₀₋₄—N(H or R₂₁₅)—SO₂—R₂₂₀,            —(CH₂)₀₋₄—N(H or R₂₁₅)—C(O)—N(R₂₁₅)₂, —(CH₂)₀₋₄—N(H or            R₂₁₅)—C(O)—R₂₂₀, —(CH₂)₀₋₄—O—C(O)-alkyl, —(CH₂)₀₋₄—O—(R₂₁₅),            —(CH₂)₀₋₄—S—(R₂₁₅), —(CH₂)₀₋₄—O-alkyl optionally substituted            with at least one halogen, and -adamantane;            -   wherein each aryl and heteroaryl group included within                R₂₀₀ is optionally substituted with at least one group                independently selected from R₂₀₅, R₂₁₀, and alkyl                (optionally substituted with at least one group                independently selected from R₂₀₅ and R₂₁₀);            -   wherein each cycloalkyl or heterocycloalkyl group                included within R₂₀₀ is optionally substituted with at                least one group independently selected from R₂₁₀;    -   R₂₀₅ at each occurrence is independently selected from -alkyl,        -haloalkoxy, —(CH₂)₀₋₃-cycloalkyl, -halogen, —(CH₂)₀₋₆—OH,        —O-aryl, —OH, —SH, —(CH₂)₀₋₄—C(O)H, —(CH₂)₀₋₆—CN,        —(CH₂)₀₋₆—C(O)—NR₂₃₅R₂₄₀, —(CH₂)₀₋₆—C(O)—R₂₃₅, —(CH₂)₀₋₄—N(H or        R₂₁₅)—SO₂—R₂₃₅, —OCF₃, —CF₃, -alkoxy, -alkoxycarbonyl, and        —NR₂₃₅R₂₄₀; R₂₁₀ at each occurrence is independently selected        from —(CH₂)₀₋₄—OH, —(CH₂)₀₋₄—CN, —(CH₂)₀₋₄—C(O)H, -alkyl        optionally substituted with at least one group independently        selected from R₂₀₅, -alkanoyl, —S-alkyl; —S(O)₂-alkyl, -halogen,        -alkoxy, -haloalkoxy, —NR₂₂₀R₂₂₅, -cycloalkyl optionally        substituted with at least one group independently selected from        R₂₀₅, -heterocycloalkyl, -heteroaryl, —(CH₂)₀₋₄—NR₂₃₅R₂₄₀,        —(CH₂)₀₋₄—NR₂₃₅(alkoxy), —(CH₂)₀₋₄—S—(R₂₁₅),        —(CH₂)₀₋₄—NR₂₃₅—C(O)H, —(CH₂)₀₋₄—NR₂₃₅—C(O)-(alkoxy),        —(CH₂)₀₋₄—NR₂₃₅—C(O)—R₂₄₀, —C(O)—NHR₂₁₅, —C(O)-alkyl,        —C(O)—NR₂₃₅R₂₄₀, and —S(O)₂—NR₂₃₅R₂₄₀;    -   R₂₁₅ at each occurrence is independently selected from -alkyl,        —(CH₂)₀₋₂-aryl, —(CH₂)₀₋₂-cycloalkyl, —(CH₂)₀₋₂-heteroaryl,        —(CH₂)₀₋₂-heterocycloalkyl, and        -   —CO₂—CH₂-aryl; wherein the aryl group included within R₂₁₅            is optionally substituted with at least one group            independently selected from R₂₀₅ and R₂₁₀, and wherein the            heterocycloalkyl and heteroaryl groups included within R₂₁₅            are optionally substituted with at least one group            independently selected from R₂₁₀;    -   R₂₂₀ and R₂₂₅ at each occurrence are independently selected from        —H, alkyl, —(CH₂)₀₋₄—C(O)H, alkylhydroxyl, alkoxycarbonyl,        alkylamino, —S(O)₂-alkyl, alkanoyl (optionally substituted with        at least one halogen), —C(O)—NH₂, —C(O)—NH(alkyl),        —C(O)—N(alkyl)(alkyl), haloalkyl, —(CH₂)₀₋₂-cycloalkyl,        -(alkyl)-O-(alkyl), aryl, heteroaryl, and heterocycloalkyl;        -   wherein the aryl, heteroaryl, cycloalkyl and            heterocycloalkyl groups included within R₂₂₀ and R₂₂₅ are            each optionally substituted with at least one group            independently selected from R₂₇₀;    -   R₂₇₀ at each occurrence is independently selected from —R₂₀₅,        alkyl (optionally substituted with at least one group        independently selected from R₂₀₅), aryl, halogen, alkoxy,        haloalkoxy, —NR₂₃₅R₂₄₀, —OH, —CN, cycloalkyl (optionally        substituted with at least one group independently selected from        R₂₀₅), —C(O)-alkyl, —S(O)₂—NR₂₃₅R₂₄₀, —C(O)—NR₂₃₅R₂₄₀,        —S(O)₂-alkyl, and —(CH₂)₀₋₄—C(O)H;    -   R₂₃₅ and R₂₄₀ at each occurrence are independently selected from        —H, —OH, —CF₃, —OCF₃, —OCH₃, —NHCH₃, —N(CH₃)₂, —(CH₂)₀₋₄—C(O)(H        or alkyl), alkyl, alkanoyl, —SO₂-alkyl, and aryl.

Exemplary R₆₀₀ substituents of monocyclic, bicyclic, or tricyclicheteroaryls include Benzo[4,5]thieno[3,2-d]pyrimidin-4-yl,4,6-Diamino-[1,3,5]triazin-2-yl, 3-nitro-pyridin-2-yl,5-trifluoromethyl-pyridin-2-yl, 8-trifluoromethyl-quinolin-4-yl,4-trifluoromethyl-pyrimidin-2-yl, 2-phenyl-quinazolin-4-yl,6-Chloro-pyrazin-2-yl, pyrimidin-2-yl, quinolin-2-yl,3-Chloro-pyrazin-2-yl, 6-Chloro-2,5-diphenyl-pyrimidin-4-yl,3-Chloro-quinoxalin-2-yl, 5-ethyl-pyrimidin-2-yl,6-Chloro-2-methylsulfanyl-5-phenyl-pyrimidin-4-yl, quinolin-4-yl,3-ethoxycarbonyl-pyridin-2yl, 5-Cyano-pyridin-2-yl,2-phenyl-quinolin-4-yl, 7H-purin-6-yl, 3-Cyano-pyridin-2-yl,4,6-dimethoxy-[1,3,5]triazin-2-yl, 3-Cyano-pyrazin-2-yl,9-(tetrahydro-pyran-2-yl)-9H-purin-6-yl, 2-Chloro-7H-purin-6-yl,2-Amino-6-chloro-pyrimidin-4-yl, 2-Chloro-6-methyl-pyrimidin-4-yl,2-Amino-6-methyl-pyrimidin-4-yl, 4-Chloro-pyrimidin-2-yl,2-Amino-7H-purin-6-yl, 4-trifluoromethyl-pyrimidin-2-yl, and the like.

Exemplary R₂ substituents include3-Allyl-5-benzyl-2-oxo-imidazolidin-1-yl,6-Benzyl-3,3-dimethyl-2-oxo-piperazin-1-yl,3-Allyl-5-benzyl-2-oxo-pyrrolidin-1-yl,5-Benzyl-3-isobutyl-2-oxo-imidazolidin-1-yl,3-Benzyl-5-methyl-1,1-dioxo-1λ⁶-[1,2,5]thiadiazolidin-2-yl,3-Benzyl-1,1-dioxo-1λ⁶-isothiazolidin-2-yl,2-Benzyl-5-oxo-pyrrolidin-1-yl, 5-Benzyl-3-ethyl-2-oxo-pyrrolidin-1-yl,3-Amino-5-benzyl-2-oxo-pyrrolidin-1-yl,3-Acetylamino-5-benzyl-2-oxo-pyrrolidin-1-yl,5-Benzyl-3-[1,3]dioxolan-4-ylmethyl-2-oxo-pyrrolidin-1-yl,3-Benzyl-5-oxo-morpholin-4-yl, 2-Benzyl-6-oxo-piperazin-1-yl,8-Benzyl-6-methyl-10-oxo-6,9-diaza-spiro[4.5]dec-9-yl,5-Benzyl-3-furan-2-ylmethylene-2-oxo-pyrrolidin-1-yl,3-acetylamino-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-acetylamino-3-(cyclopropylmethyl)-2-oxo-pyrrolidin-1-yl,3-(2-amino-5-carboxypentanoylamino)-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-(2-methoxy-acetylamino)-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-ethoxycarbonylamino-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-ethylureido-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl, and3-hydroxypropionylamino-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl.

In an embodiment, R₁ is selected from 3-allyloxy-5-fluoro-benzyl,3-benzyloxy-5-fluoro-benzyl, 3-propyl-thiophen-2-yl-methyl,3,5-difluoro-2-propylamino-benzyl, 2-ethylamino-3,5-difluoro-benzyl,2-hydroxy-5-methyl-benzamide,3-fluoro-5-[2-(2-methoxy-ethoxy)-ethoxy]-benzyl,3-fluoro-5-heptyloxy-benzyl, and 3-fluoro-5-hexyloxy-benzyl.

In another embodiment, R₁ is selected from a —CH₂-aryl, wherein the arylring is optionally substituted with at least one group independentlyselected from halogen, —C₁-C₂ alkyl, —C1-C₂ alkoxy, and —OH.

In another embodiment, R₁ is selected from 3-Allyloxy-5-fluoro-benzyl,3-Benzyloxy-5-fluoro-benzyl, 4-hydroxy-benzyl, 3-hydroxy-benzyl,3-propyl-thiophen-2-yl-methyl, 3,5-difluoro-2-propylamino-benzyl,5-chloro-thiophen-2-yl-methyl, 5-chloro-3-ethyl-thiophen-2-yl-methyl,3,5-difluoro-2-hydroxy-benzyl, 2-ethylamino-3,5-difluoro-benzyl,piperidin-4-yl-methyl, 2-oxo-piperidin-4-yl-methyl,2-oxo-1,2-dihydro-pyridin-4-yl-methyl,5-hydroxy-6-oxo-6H-pyran-2-yl-methyl, 2-Hydroxy-5-methyl-benzamide,3,5-Difluoro-4-hydroxy-benzyl, 3,5-Difluoro-benzyl,3-Fluoro-4-hydroxy-benzyl,3-Fluoro-5-[2-(2-methoxy-ethoxy)-ethoxy]-benzyl,3-Fluoro-5-heptyloxy-benzyl, 3-Fluoro-5-hexyloxy-benzyl,3-Fluoro-5-hydroxy-benzyl, 3-Fluoro-benzyl, and the like.

Exemplary R₂ substituents include3-Allyl-5-benzyl-2-oxo-imidazolidin-1-yl,6-Benzyl-3,3-dimethyl-2-oxo-piperazin-1-yl,3-Allyl-5-benzyl-2-oxo-pyrrolidin-1-yl,5-Benzyl-3-isobutyl-2-oxo-imidazolidin-1-yl,3-Benzyl-5-methyl-1,1-dioxo-1λ⁶-[1,2,5]thiadiazolidin-2-yl,3-Benzyl-1,1-dioxo-1λ⁶-isothiazolidin-2-yl,2-Benzyl-5-oxo-pyrrolidin-1-yl, 5-Benzyl-3-ethyl-2-oxo-pyrrolidin-1-yl,3-Amino-5-benzyl-2-oxo-pyrrolidin-1-yl,3-Acetylamino-5-benzyl-2-oxo-pyrrolidin-1-yl,5-Benzyl-3-[1,3]dioxolan-4-ylmethyl-2-oxo-pyrrolidin-1-yl,3-Benzyl-5-oxo-morpholin-4-yl, 2-Benzyl-6-oxo-piperazin-1-yl,8-Benzyl-6-methyl-10-oxo-6,9-diaza-spiro[4.5]dec-9-yl,5-Benzyl-3-furan-2-ylmethylene-2-oxo-pyrrolidin-1-yl,3-acetylamino-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-acetylamino-3-(cyclopropylmethyl)-2-oxo-pyrrolidin-1-yl,3-(2-amino-5-carboxypentanoylamino)-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-(2-methoxy-acetylamino)-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-ethoxycarbonylamino-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-ethylureido-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl,3-hydroxypropionylamino-3-(sec-butyl)-2-oxo-pyrrolidin-1-yl, and thelike.

In another embodiment, R₂ is selected from hydrogen,3-Bromo-[1,2,4]thiadiazol-5-ylamino, [1,2,4]thiadiazol-5-ylamino,4-Chloro-[1,2,5]thiadiazol-3-ylamino, [1,2,5]thiadiazol-3-ylamino,thiazol-2-ylamino, 5-Bromo-[1,3,4]thiadiazol-2-ylamino,[1,3,4]thiadiazol-2-ylamino, 5-Amino-[1,3,4]thiadiazol-2-ylamino,2-Bromo-thiazol-5-ylamino, thiazol-5-ylamino,5-trifluoromethyl-[1,3,4]thiadiazol-2-ylamino,5-trifluoromethyl-[1,3,4]oxadiazol-2-ylamino,5-Amino-[1,3,4]oxadiazol-2-ylamino,1-trityl-1H-[1,2,4]triazol-3-ylamino, 1H-[1,2,4]triazol-3-ylamino,oxazol-2-ylamino, 5-Bromo-2-trityl-2H-[1,2,3]triazol-4-ylamino,2-trityl-2H-[1,2,3]triazol-4-ylamino,5-Bromo-2H-[1,2,3]triazol-4-ylamino, 2H-[1,2,3]triazol-4-ylamino,thiophen-2-ylamino, 3-methyl-5-nitro-3H-imidazol-4-ylamino,4-Cyano-5-phenyl-isothiazol-3-ylamino,4-phenyl-[1,2,5]thiadiazol-3-ylamino, 3,4-dioxo-cyclobut-1-enylamino,2-methoxy-3,4-dioxo-cyclobut-1-enylamino, and2-methylamino-3,4-dioxo-cyclobut-1-enylamino, and the like.

In another embodiment, R_(C) is selected from

wherein A₅ is —C(═N—R₂₃₀) and A₁, A₂, A₃, A₄, R_(X) and R₂₃₀ are definedabove.

In another embodiment, A₅ is selected from —C(═N—OH)—, —C(═N—O—CH₃)—,—C(═N—O—CH₂CH₃)—, —C(═N—O—CH₂CH₂OH)—, —C(═N—O—CH₂CH₂NH₂)—,—C(═N—NHCH₃)—, and —C(═N—CN)—, and A₁, A₂, A₃, and A₄ are —CH₂—.

In another embodiment, A₅ is selected from —C(═N—OH)—, —C(═N—O—CH₃)—,—C(═N—O—CH₂CH₃)—, —C(═N—O—CH₂CH₂OH)—, —C(═N—O—CH₂CH₂NH₂)—,—C(═N—NHCH₃)—, and —C(═N—CN)—.

In another embodiment, R_(C) is selected from1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-(2-amino-ethoxyimino)-cyclohexyl,5-(3-tert-Butyl-phenyl)-2-hydroxyimino-hexahydro-pyrimidin-5-yl,1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexyl,5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl,5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-benzo[c]isoxazol-5-yl,1-(Acrylic acid methyl ester)-4-(tert-Butyl-phenyl)-cyclohexane-4-yl,1-(Acrylamide)-4-(tert-Butyl-phenyl)-cyclohexane-4-yl,1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethylidene)-cyclohex-1-yl,1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohex-1-yl,1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohex-1-yl,4-methoxyimino-1-(3-thiophen-3-yl-phenyl)-cyclohexyl,1-(3-furan-3-yl-phenyl)-4-methoxyimino-cyclohexyl,4-methoxyimino-1-[3-(1H-pyrrol-2-yl)-phenyl]-cyclohexyl,4-methoxyimino-1-(3-pyridin-4-yl-phenyl)-cyclohexyl,4-methoxyimino-1-(3-pyrimidin-5-yl-phenyl)-cyclohexyl,4-methoxyimino-1-(3-pyrazol-1-yl-phenyl)-cyclohexyl,2-Acetyl-5-(3-tert-butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl,1-(3-tert-Butyl-phenyl)-4-methylene-cyclohexyl, ethyl2-(4-(3-tert-butylphenyl)cyclohexylidene)acetate, and the like.

In another embodiment, R_(X) is selected from3-(1,1-dimethyl-propyl)-phenyl, 3-(1-ethyl-propyl)-phenyl,3-(1H-pyrrol-2-yl)-phenyl, 3-(1-hydroxy-1-methyl-ethyl)-phenyl,3-(1-methyl-1H-imidazol-2-yl)-phenyl, 3-(1-methyl-cyclopropyl)-phenyl,3-(2,2-dimethyl-propyl)-phenyl, 3-(2,5-dihydro-1H-pyrrol-2-yl)-phenyl,3-(2-Chloro-thiophen-3-yl)-phenyl, 3-(2-Cyano-thiophen-3-yl)-phenyl,3-(2-fluoro-benzyl)-phenyl, 3-(3,5-dimethyl-3H-pyrazol-4-yl)-phenyl,3-(3,6-dimethyl-pyrazin-2-yl)-phenyl, 3-(3-Cyano-pyrazin-2-yl)-phenyl,3-(3-formyl-furan-2-yl)-phenyl, 3-(3H-[1,2,3]triazol-4-yl)-phenyl,3-(3H-imidazol-4-yl)-phenyl, 3-(3-methyl-butyl)-phenyl,3-(3-methyl-pyridin-2-yl)-phenyl, 3-(3-methyl-thiophen-2-yl)-phenyl,3-(4-Cyano-pyridin-2-yl)-phenyl, 3-(4-fluoro-benzyl)-phenyl,3-(4H-[1,2,4]triazol-3-yl)-phenyl, 3-(4-methyl-thiophen-2-yl)-phenyl,3-(5-Acetyl-thiophen-2-yl)-phenyl, 3-(5-Acetyl-thiophen-3-yl)-phenyl,3-(5-formyl-thiophen-2-yl)-phenyl, 3-(5-oxo-pyrrolidin-2-yl)-phenyl,3-(6-methyl-pyridazin-3-yl)-phenyl, 3-(6-methyl-pyridin-2-yl)-phenyl,3-(Cyano-dimethyl-methyl)-phenyl,3-[1-(2-tert-Butyl-pyrimidin-4-yl)-cyclohexylamino,3-[1,2,3]triazol-1-yl-phenyl, 3-[1,2,4]oxadiazol-3-yl-phenyl,3-[1,2,4]oxadiazol-5-yl-phenyl, 3-[1,2,4]thiadiazol-3-yl-phenyl,3-[1,2,4]thiadiazol-5-yl-phenyl, 3-[1,2,4]triazol-4-yl-phenyl,3-Acetyl-5-tert-butyl-phenyl, 3′-Acetylamino-biphenyl-3-yl,3-Adamantan-2-yl-phenyl, 3-Bromo-[1,2,4]thiadiazol-5-yl)-phenyl,3-Bromo-5-tert-butyl-phenyl, 3-Cyano-phenyl, 3-Cyclobutyl-phenyl,3-Cyclopentyl-phenyl, 3-Cyclopropyl-phenyl, 3-ethyl-phenyl,3-ethynyl-phenyl, 3-fluoro-5-(2-hydroxy-1,1-dimethyl-ethyl)-phenyl,3-furan-3-yl-phenyl, 3-imidazol-1-yl-phenyl, 3-isobutyl-phenyl,3-isopropyl-phenyl, 3-isoxazol-3-yl-phenyl, 3-isoxazol-4-yl-phenyl,3-isoxazol-5-yl-phenyl, 3-pent-4-enyl-phenyl, 3-pentyl-phenyl,3-Phenyl-propionic acid ethyl ester, 3-pyrazin-2-yl-phenyl,3-pyridin-2-yl-phenyl, 3-pyrrolidin-2-yl-phenyl, 3-sec-Butyl-phenyl,3-tert-Butyl-4-chloro-phenyl, 3-tert-Butyl-4-cyano-phenyl,3-tert-Butyl-4-ethyl-phenyl, 3-tert-Butyl-4-methyl-phenyl,3-tert-Butyl-4-trifluoromethyl-phenyl, 3-tert-Butyl-5-chloro-phenyl,3-tert-Butyl-5-cyano-phenyl, 3-tert-Butyl-5-ethyl-phenyl,3-tert-Butyl-5-fluoro-phenyl, 3-tert-Butyl-5-methyl-phenyl,3-tert-Butyl-5-trifluoromethyl-phenyl, 3-tert-Butyl-phenyl,3-thiazol-2-yl-phenyl, 3-thiazol-4-yl-phenyl, 3-thiophen-3-yl-phenyl,3-trifluoromethyl-phenyl, 4-Acetyl-3-tert-butyl-phenyl,4-tert-Butyl-pyridin-2-yl, 4-tert-Butyl-pyrimidin-2-yl,5-tert-Butyl-pyridazin-3-yl, and 6-tert-Butyl-pyridazin-4-yl,6-tert-Butyl-pyrimidin-4-yl, and the like.

In another embodiment, R_(X) is 3-tert-Butyl-phenyl.

In another embodiment, the present invention provides a method ofpreventing or treating at least one condition that benefits frominhibition of at least one aspartyl-protease, comprising administeringto a host a composition comprising a therapeutically effective amount ofat least one compound of the formula,

or pharmaceutically acceptable salts thereof, wherein R₁, R₂, and R_(C)are as defined below and R₀ is selected from —CH(alkyl)-, —C(alkyl)₂-,—CH(cycloalkyl)-, —C(alkyl)(cycloalkyl)-, and —C(cycloalkyl)₂-.

In another embodiment, the present invention encompasses compounds offormula (I) wherein the hydroxyl substituent alpha to the —(CHR₁)—group, as shown in formula (I), may optionally be replaced by —NH₂,—NH(R₇₀₀), —N(R₇₀₀)(R₇₀₀), —SH, and —SR₇₀₀, wherein R₇₀₀ is alkyloptionally substituted with at least one group independently selectedfrom R₂₀₀, R₂₀₅, R₂₁₀, R₂₁₅, R₂₂₀, and R₂₂₅.

Among the compounds of formula (I), or pharmaceutically acceptable saltsthereof, examples include4-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[4-(2-Amino-ethoxyimino)-1-(3-tert-butyl-phenyl)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[5-(3-tert-Butyl-phenyl)-2-hydroxyimino-hexahydro-pyrimidin-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-benzo[c]isoxazol-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methylcarbamoylmethylene-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,{4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-methylcarbamoyl-butylamino]-cyclohexylidene}-aceticacid methyl ester,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethylidene)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[4-(2-Amino-ethoxyimino)-1-(3-tert-butyl-phenyl)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[5-(3-tert-Butyl-phenyl)-2-hydroxyimino-hexahydro-pyrimidin-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-benzo[c]isoxazol-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methylcarbamoylmethylene-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,{4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-phenylcarbamoyl-butylamino]-cyclohexylidene}-aceticacid methyl ester,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethylidene)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-(1H-imidazol-2-yl)-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[3-(3,5-difluoro-phenoxy)-2-hydroxy-propylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[3-(3,5-difluoro-benzenesulfonyl)-2-hydroxy-propylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-4-oxo-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-pentylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-pentylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-tetrazol-1-yl-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-(5-trifluoromethyl-[1,3,4]oxadiazol-2-yl)-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-([1,2,4]thiadiazol-5-ylamino)-butylamino]-cyclohexanoneoxime,3-[3-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propylamino]-4-methylamino-cyclobut-3-ene-1,2-dione,3-[3-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propylamino]-4-methoxy-cyclobut-3-ene-1,2-dione,4-(3-tert-Butyl-phenyl)-4-[2-hydroxy-4-(3-propyl-thiophen-2-yl)-3-([1,2,4]thiadiazol-5-ylamino)-butylamino]-cyclohexanoneoxime,1-(5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino)-4-(3,5-difluorophenyl)butan-2-ol,1-(5-(3-tert-butylphenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamino)-4-(3,5-difluorophenyl)butan-2-ol,and the like.

The present invention encompasses methods of treatment using compoundswith structural characteristics designed for interacting with theirtarget molecules. Such characteristics include at least one moietycapable of interacting with at least one subsite of beta-secretase. Suchcharacteristics also include at least one moiety capable of enhancingthe interaction between the target and at least one subsite ofbeta-secretase.

It is preferred that the compounds of formula (I) are efficacious. Forexample, it is preferred that the compounds of formula (I) decrease thelevel of beta-secretase using low dosages of the compounds. Preferably,the compounds of formula (I) decrease the level of A-beta by at least10% using dosages of about 100 mg/kg. It is more preferred that thecompounds of formula (I) decrease the level of A-beta by at least 10%using dosages of less than 100 mg/kg. It is also more preferred that thecompounds of formula (I) decrease the level of A-beta by greater than10% using dosages of about 100 mg/kg. It is most preferred that thecompounds of formula (I) decrease the level of A-beta by greater than10% using dosages of less than 100 mg/kg.

In an embodiment, the host is a cell.

In another embodiment, the host is an animal.

In another embodiment, the host is human.

In another embodiment, at least one compound of formula (I) isadministered in combination with a pharmaceutically acceptable carrieror diluent.

In another embodiment, the pharmaceutical compositions comprisingcompounds of formula (I) can be used to treat a wide variety ofdisorders or conditions including Alzheimer's disease, Down's syndromeor Trisomy 21 (including mild cognitive impairment (MCI) Down'ssyndrome), hereditary cerebral hemorrhage with amyloidosis of the Dutchtype, chronic inflammation due to amyloidosis, prion diseases (includingCreutzfeldt-Jakob disease, Gerstmann-Straussler syndrome, kuru scrapie,and animal scrapie), Familial Amyloidotic Polyneuropathy, cerebralamyloid angiopathy, other degenerative dementias including dementias ofmixed vascular and degenerative origin, dementia associated withParkinson's disease, dementia associated with progressive supranuclearpalsy and dementia associated with cortical basal degeneration, diffuseLewy body type of Alzheimer's disease, and frontotemporal dementias withparkinsonism (FTDP).

In another embodiment, the condition is Alzheimer's disease.

In another embodiment, the condition is dementia.

When treating or preventing these diseases, the methods of the presentinvention can either employ the compounds of formula (I) individually orin combination, as is best for the patient.

In treating a patient displaying any of the conditions discussed above,a physician may employ a compound of formula (I) immediately andcontinue administration indefinitely, as needed. In treating patientswho are not diagnosed as having Alzheimer's disease, but who arebelieved to be at substantial risk for it, the physician may starttreatment when the patient first experiences early pre-Alzheimer'ssymptoms, such as memory or cognitive problems associated with aging. Inaddition, there are some patients who may be determined to be at riskfor developing Alzheimer's disease through the detection of a geneticmarker such as APOE4 or other biological indicators that are predictivefor Alzheimer's disease and related conditions. In these situations,even though the patient does not have symptoms of the disease orcondition, administration of the compounds of formula (I) may be startedbefore symptoms appear, and treatment may be continued indefinitely toprevent or delay the onset of the disease. Similar protocols areprovided for other diseases and conditions associated with amyloidosis,such as those characterized by dementia.

In an embodiment, the methods of preventing or treating at least onecondition associated with amyloidosis, comprising administering to ahost a composition comprising a therapeutically effective amount of atleast one compound of formula (I), which may include beta-secretasecomplexed with at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C) areas previously defined.

One embodiment of the present invention provides a method of preventingor treating the onset of Alzheimer's disease comprising administering toa patient a therapeutically effective amount of at least one compound offormula (I), or at least one pharmaceutically acceptable salt thereof,wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofpreventing or treating the onset of dementia comprising administering toa patient a therapeutically effective amount of at least one compound offormula (I), or at least one pharmaceutically acceptable salt thereof,wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofpreventing or treating at least one condition associated withamyloidosis by administering to a host an effective amount of at leastone compound of formula (I), or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofpreventing or treating Alzheimer's disease by administering to a host aneffective amount of at least one compound of formula (I), or at leastone pharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C)are as previously defined.

Another embodiment of the present invention provides a method ofpreventing or treating dementia by administering to a host an effectiveamount of at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C) areas previously defined.

Another embodiment of the present invention provides a method ofinhibiting beta-secretase activity in a cell. This method comprisesadministering to the cell an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofinhibiting beta-secretase activity in a host. This method comprisesadministering to the host an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofinhibiting beta-secretase activity in a host. This method comprisesadministering to the host an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are as previously defined, andwherein the host is a human.

Another embodiment of the present invention provides methods ofaffecting beta-secretase-mediated cleavage of amyloid precursor proteinin a patient, comprising administering a therapeutically effectiveamount of at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C) areas previously defined.

Another embodiment of the present invention provides a method ofinhibiting cleavage of amyloid precursor protein at a site betweenMet596 and Asp597 (numbered for the APP-695 amino acid isotype), or at acorresponding site of an isotype or mutant thereof, comprisingadministering a therapeutically effective amount of at least onecompound of formula (I), or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofinhibiting cleavage of amyloid precursor protein or mutant thereof at asite between amino acids, comprising administering a therapeuticallyeffective amount of at least one compound of formula (I), or at leastone pharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C)are as previously defined, and wherein the site between amino acidscorresponds to between Met652 and Asp653 (numbered for the APP-751isotype), between Met671 and Asp672 (numbered for the APP-770 isotype),between Leu596 and Asp597 of the APP-695 Swedish Mutation, betweenLeu652 and Asp653 of the APP-751 Swedish Mutation, or between Leu671 andAsp672 of the APP-770 Swedish Mutation.

Another embodiment of the present invention provides a method ofinhibiting production of A-beta, comprising administering to a patient atherapeutically effective amount of at least one compound of formula(I), or at least one pharmaceutically acceptable salt thereof, whereinR₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofpreventing or treating deposition of A-beta, comprising administering atherapeutically effective amount of at least one compound of formula(I), or at least one pharmaceutically acceptable salt thereof, whereinR₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofpreventing, delaying, halting, or reversing a disease characterized byA-beta deposits or plaques, comprising administering a therapeuticallyeffective amount of at least one compound of formula (I), or at leastone pharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C)are as previously defined.

In one embodiment the A-beta deposits or plaques are in a human brain.

Another embodiment of the present invention provides a method ofpreventing, delaying, halting, or reversing a condition associated witha pathological form of A-beta in a host comprising administering to apatient in need thereof an effective amount of at least one compound offormula (I), or at least one pharmaceutically acceptable salt thereof,wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides a method ofinhibiting the activity of at least one aspartyl protease in a patientin need thereof, comprising administering a therapeutically effectiveamount of at least one compound of formula (I), or at least onepharmaceutically acceptable salt thereof to the patient, wherein R₁, R₂,and R_(C) are as previously defined.

In one embodiment, the at least one aspartyl protease is beta-secretase.

Another embodiment of the present invention provides a method ofinteracting an inhibitor with beta-secretase, comprising administeringto a patient in need thereof a therapeutically effective amount of atleast one compound of formula (I), or at least one pharmaceuticallyacceptable salt thereof, wherein R₁, R₂, and R_(C) are as previouslydefined, and wherein the at least one compound interacts with at leastone beta-secretase subsite such as S1, S1′, or S2′.

Another embodiment provides a method of selecting compounds of formula(I) wherein the pharmacokinetic parameters are adjusted for a anincrease in desired effect (e.g., increased brain uptake).

Another embodiment provides a method of selecting at least one compoundof formula (I) wherein C_(max), T_(max), and/or half-life are adjustedto provide for maximum efficacy.

Another embodiment of the present invention provides a method oftreating a condition in a patient, comprising administering atherapeutically effective amount of at least one compound of formula(I), or at least one pharmaceutically acceptable salt, derivative orbiologically active metabolite thereof, to the patient, wherein R₁, R₂,and R_(C) are as previously defined.

In an embodiment, the condition is Alzheimer's disease.

In another embodiment, the condition is dementia.

In another embodiment, the compounds of formula (I) are administered inoral dosage form. The oral dosage forms are generally administered tothe patient 1, 2, 3, or 4 times daily. It is preferred that thecompounds be administered either three or fewer times daily, morepreferably once or twice daily. It is preferred that, whatever oraldosage form is used, it be designed so as to protect the compounds fromthe acidic environment of the stomach. Enteric coated tablets are wellknown to those skilled in the art. In addition, capsules filled withsmall spheres, each coated to be protected from the acidic stomach, arealso well known to those skilled in the art.

Therapeutically effective amounts include, for example, oraladministration from about 0.1 mg/day to about 1,000 mg/day, parenteral,sublingual, intranasal, intrathecal administration from about 0.2 mg/dayto about 100 mg/day, depot administration and implants from about 0.5mg/day to about 50 mg/day, topical administration from about 0.5 mg/dayto about 200 mg/day, and rectal administration from about 0.5 mg/day toabout 500 mg/day.

When administered orally, an administered amount therapeuticallyeffective to inhibit beta-secretase activity, to inhibit A-betaproduction, to inhibit A-beta deposition, or to treat or preventAlzheimer's disease is from about 0.1 mg/day to about 1,000 mg/day.

In various embodiments, the therapeutically effective amount may beadministered in, for example, pill, tablet, capsule, powder, gel, orelixir form, and/or combinations thereof. It is understood that, while apatient may be started at one dose or method of administration, thatdose or method of administration may vary over time as the patient'scondition changes.

Another embodiment of the present invention provides a method ofprescribing a medication for preventing, delaying, halting, or reversingat least one disorder, condition or disease associated with amyloidosis.The method includes identifying in a patient symptoms associated with atleast one disorder, condition or disease associated with amyloidosis,and prescribing at least one dosage form of at least one compound offormula (I), or at least one pharmaceutically acceptable salt, to thepatient, wherein R₁, R₂, and R_(C) are as previously defined.

Another embodiment of the present invention provides an article ofmanufacture, comprising (a) at least one dosage form of at least onecompound of formula (I), or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are as previously defined, (b) apackage insert providing that a dosage form comprising a compound offormula (I) should be administered to a patient in need of therapy forat least one disorder, condition or disease associated with amyloidosis,and (c) at least one container in which at least one dosage form of atleast one compound of formula (I) is stored.

Another embodiment provides a packaged pharmaceutical composition fortreating at least one condition related to amyloidosis, comprising (a) acontainer which holds an effective amount of at least one compound offormula (I), or at least one pharmaceutically acceptable salt thereof,and (b) instructions for using the pharmaceutical composition.

Another embodiment of the present invention provides an article ofmanufacture, comprising (a) a therapeutically effective amount of atleast one compound of formula (I), or pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are as previously defined, (b) apackage insert providing an oral dosage form should be administered to apatient in need of therapy for at least one disorder, condition ordisease associated with amyloidosis, and (c) at least one containercomprising at least one oral dosage form of at least one compound offormula (I).

Another embodiment of the present invention provides an article ofmanufacture, comprising (a) at least one oral dosage form of at leastone compound of formula (I), or at least one pharmaceutically acceptablesalt thereof, wherein R₁, R₂, and R_(C) are as previously defined, in adosage amount ranging from about 2 mg to about 1000 mg, associated with(b) a package insert providing that an oral dosage form comprising acompound of formula (I) in a dosage amount ranging from about 2 mg toabout 1000 mg should be administered to a patient in need of therapy forat least one disorder, condition or disease associated with amyloidosis,and (c) at least one container in which at least one oral dosage form ofat least one compound of formula (I) in a dosage amount ranging fromabout 2 mg to about 1000 mg is stored.

Another embodiment of the present invention provides an article ofmanufacture, comprising (a) at least one oral dosage form of at leastone compound of formula (I) in a dosage amount ranging from about 2 mgto about 1000 mg in combination with (b) at least one therapeuticallyactive agent, associated with (c) a package insert providing that anoral dosage form comprising a compound of formula (I) in a dosage amountranging from about 2 mg to about 1000 mg in combination with at leastone therapeutically active agent should be administered to a patient inneed of therapy for at least one disorder, condition or diseaseassociated with amyloidosis, and (d) at least one container in which atleast one dosage form of at least one compound of formula (I) in adosage amount ranging from about 2 mg to about 1000 mg in combinationwith a therapeutically active agent is stored.

Another embodiment of the present invention provides an article ofmanufacture, comprising (a) at least one parenteral dosage form of atleast one compound of formula (I) or at least one pharmaceuticallyacceptable salt thereof, in a dosage amount ranging from about 0.2 mg/mLto about 50 mg/mL, associated with (b) a package insert providing that aparenteral dosage form comprising a compound of formula (I) in a dosageamount ranging from about 0.2 mg/mL to about 50 mg/mL should beadministered to a patient in need of therapy for at least one disorder,condition or disease associated with amyloidosis, and (c) at least onecontainer in which at least one parenteral dosage form of at least onecompound of formula (I), or at least one pharmaceutically acceptablesalt thereof, in a dosage amount ranging from about 0.2 mg/mL to about50 mg/mL is stored.

A further embodiment of the present invention provides an article ofmanufacture comprising (a) a medicament comprising an effective amountof at least one compound of formula (I) or at least one pharmaceuticallyacceptable salt thereof, in combination with active and/or inactivepharmaceutical agents, (b) a package insert providing that an effectiveamount of at least one compound of formula (I) should be administered toa patient in need of therapy for at least one disorder, condition ordisease associated with amyloidosis, and (c) a container in which amedicament comprising an effective amount of at least one compound offormula (I) in combination with a therapeutically active and/or inactiveagent is stored.

In an embodiment, the therapeutically active agent is selected from anantioxidant, an anti-inflammatory, a gamma-secretase inhibitor, aneurotrophic agent, an acetyl cholinesterase inhibitor, a statin, anA-beta, and/or an anti-A-beta antibody.

Another embodiment of the present invention provides an article ofmanufacture comprising: (a) a medicament comprising: an effective amountof at least one compound of formula (I),

or at least one pharmaceutically acceptable salt thereof, wherein R₁,R₂, and R_(C) are defined bellow, in combination with active and/orinactive pharmaceutical agents; (b) a package insert providing that aneffective amount of at least one compound of formula (I) should beadministered to a patient in need of therapy for at least one disorder,condition or disease associated with amyloidosis; and (c) a container inwhich a medicament comprising: an effective amount of at least onecompound of formula (I) in combination with active and/or inactivepharmaceutical agents is stored.

Another embodiment of the present invention provides a kit comprising:(a) at least one dosage form of at least one compound of formula (I);and (b) at least one container in which at least one dosage form of atleast one compound of formula (I) is stored.

In an embodiment, the kit further comprises a package insert: a)containing information of the dosage amount and duration of exposure ofa dosage form containing at least one compound of formula (I), or atleast one pharmaceutically acceptable salt thereof, and b) providingthat the dosage form should be administered to a patient in need oftherapy for at least one disorder, condition or disease associated withamyloidosis.

In another embodiment, the kit further comprises at least onetherapeutically active agent.

In another embodiment of a kit, the therapeutically active agent isselected from an antioxidant, an anti-inflammatory, a gamma-secretaseinhibitor, a neurotrophic agent, an acetyl cholinesterase inhibitor, astatin, an A-beta, and an anti-A-beta antibody.

A further embodiment of the present invention provides method ofpreventing or treating at least one condition associated withamyloidosis, comprising: administering to a host a compositioncomprising a therapeutically effective amount of at least one selectivebeta-secretase inhibitor of formula (I), or at least onepharmaceutically acceptable salt thereof, further comprising acomposition including beta-secretase complexed with at least onecompound of formula (I), wherein R₁, R₂, and R_(C) are defined bellow,or pharmaceutically acceptable salt thereof.

Another embodiment of the present invention provides a method ofproducing a beta-secretase complex comprising exposing beta-secretase toa compound of formula (I), or at least one pharmaceutically acceptablesalt thereof, in a reaction mixture under conditions suitable for theproduction of the complex.

Another embodiment of the present invention provides a manufacture of amedicament for preventing, delaying, halting, or reversing Alzheimer'sdisease, comprising adding an effective amount of at least one compoundof formula (I), or at least one pharmaceutically acceptable saltthereof, wherein R₁, R₂, and R_(C) are defined bellow, to apharmaceutically acceptable carrier.

Another embodiment of the present invention provides a method ofselecting a beta-secretase inhibitor comprising targeting at least onemoiety of at least one formula (I) compound, or at least onepharmaceutically acceptable salt thereof, to interact with at least onebeta-secretase subsite such as but not limited to S1, S1′, or S2′.

The methods of treatment described herein include administering thecompounds of formula (I) orally, parenterally (via intravenous injection(IV), intramuscular injection (IM), depo-IM, subcutaneous injection (SCor SQ), or depo-SQ), sublingually, intranasally (inhalation),intrathecally, topically, or rectally. Dosage forms known to thoseskilled in the art are suitable for delivery of the compounds of formula(I).

In treating or preventing the above diseases, the compounds of formula(I) are administered using a therapeutically effective amount. Thetherapeutically effective amount will vary depending on the particularcompound used and the route of administration, as is known to thoseskilled in the art.

The compositions are preferably formulated as suitable pharmaceuticalpreparations, such as for example, pill, tablet, capsule, powder, gel,or elixir form, and/or combinations thereof, for oral administration orin sterile solutions or suspensions for parenteral administration.Typically the compounds described above are formulated intopharmaceutical compositions using techniques and/or procedures wellknown in the art.

For example, a therapeutically effective amount of a compound or mixtureof compounds of formula (I), or a physiologically acceptable salt iscombined with a physiologically acceptable vehicle, carrier, binder,preservative, stabilizer, flavor, and the like, in a unit dosage form ascalled for by accepted pharmaceutical practice and is defined herein.The amount of active substance in those compositions or preparations issuch that a suitable dosage in the range indicated is obtained. Thecompound concentration is effective for delivery of an amount uponadministration that lessens or ameliorates at least one symptom of thedisorder for which the compound is administered. For example, thecompositions can be formulated in a unit dosage form, each dosagecontaining from about 2 mg to about 1000 mg.

The active ingredient may be administered in a single dose, or may bedivided into a number of smaller doses to be administered at intervalsof time. It is understood that the precise dosage and duration oftreatment is a function of the disease or condition being treated andmay be determined empirically using known testing protocols or byextrapolation from in vivo or in vitro test data. It is to be noted thatconcentrations and dosage values may vary with the severity of thecondition to be alleviated. It is also to be understood that the precisedosage and treatment regimens may be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth-herein are exemplary only andare not intended to limit the scope or practice of the claimedcompositions. A dosage and/or treatment method for any particularpatient also may depend on, for example, the age, weight, sex, diet,and/or health of the patient, the time of administration, and/or anyrelevant drug combinations or interactions.

To prepare compositions to be employed in the methods of treatment, atleast one compound of formula (I) or at least one pharmaceuticallyacceptable salt thereof, wherein R₁, R₂, and R_(C) are defined below, ismixed with a suitable pharmaceutically acceptable carrier. Upon mixingor addition of the compound(s), the resulting mixture may be a solution,suspension, emulsion, or the like. Liposomal suspensions may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known to those skilled in the art. The form of theresulting mixture depends upon a number of factors, including theintended mode of administration and the solubility of the compound inthe selected carrier or vehicle. An effective concentration issufficient for lessening or ameliorating at least one symptom of thedisease, disorder, or condition treated and may be empiricallydetermined.

Pharmaceutical carriers or vehicles suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration. Additionally, the active materials can also be mixedwith other active materials that do not impair the desired action, orwith materials that supplement the desired action, or have anotheraction. For example, the compounds of formula (I) may be formulated asthe sole pharmaceutically active ingredient in the composition or may becombined with other active ingredients.

Where the compounds exhibit insufficient solubility, methods forsolubilizing may be used. Such methods are known and include, forexample, using co-solvents (such as dimethylsulfoxide (DMSO)), usingsurfactants (such as Tween®), and/or dissolution in aqueous sodiumbicarbonate. Derivatives of the compounds, such as salts, metabolites,and/or pro-drugs, may also be used in formulating effectivepharmaceutical compositions. Such derivatives may improve thepharmacokinetic properties of treatment administered.

A kit may include a plurality of containers, each container holding atleast one unit dose of the compound of the present invention. Thecontainers are preferably adapted for the desired mode ofadministration, including, for example, pill, tablet, capsule, powder,gel or gel capsule, sustained-release capsule, or elixir form, and/orcombinations thereof and the like for oral administration, depotproducts, pre-filled syringes, ampoules, vials, and the like forparenteral administration, and patches, medipads, creams, and the likefor topical administration.

The tablets, pills, capsules, troches, and the like may contain a binder(e.g., gum tragacanth, acacia, corn starch, gelatin, and the like); avehicle (e.g., microcrystalline cellulose, starch, lactose, and thelike); a disintegrating agent (e.g., alginic acid, corn starch, and thelike); a lubricant (e.g., magnesium stearate, and the like); a gildant(e.g., colloidal silicon dioxide, and the like); a sweetening agent(e.g., sucrose, saccharin, and the like); a flavoring agent (e.g.,peppermint, methyl salicylate, and the like); or fruit flavoring;compounds of a similar nature, and/or mixtures thereof.

When the dosage unit form is a capsule, it can contain, in addition tomaterial described above, a liquid carrier such as a fatty oil.Additionally, dosage unit forms can contain various other materials,which modify the physical form of the dosage unit, for example, coatingsof sugar or other enteric agents. A method of treatment can alsoadminister the compound as a component of an elixir, suspension, syrup,wafer, chewing gum, or the like. A syrup may contain, in addition to theactive compounds, sucrose as a sweetening agent, flavors, preservatives,dyes and/or colorings.

The methods of treatment may employ at least one carrier that protectsthe compound against rapid elimination from the body, such astime-release formulations or coatings. Such carriers include controlledrelease formulations, such as, for example, implants ormicroencapsulated delivery systems, and the like or biodegradable,biocompatible polymers such as collagen, ethylene vinyl acetate,polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, andthe like. Methods for preparation of such formulations are known tothose in the art.

When orally administered, the compounds of the present invention can beadministered in usual dosage forms for oral administration as is wellknown to those skilled in the art. These dosage forms include the usualsolid unit dosage forms of tablets and capsules as well as liquid dosageforms such as solutions, suspensions, and elixirs. When solid dosageforms are used, it is preferred that they be of the sustained releasetype so that the compounds of the present invention need to beadministered only once or twice daily. When liquid oral dosage forms areused, it is preferred that they be of about 10 mL to about 30 mL each.Multiple doses may be administered daily.

The methods of treatment may also employ a mixture of the activematerials and other active or inactive materials that do not impair thedesired action, or with materials that supplement the desired action.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include a sterile diluent (e.g., water forinjection, saline solution, fixed oil, and the like); a naturallyoccurring vegetable oil (e.g., sesame oil, coconut oil, peanut oil,cottonseed oil, and the like); a synthetic fatty vehicle (e.g., ethyloleate, polyethylene glycol, glycerine, propylene glycol, and the like,including other synthetic solvents); antimicrobial agents (e.g., benzylalcohol, methyl parabens, and the like); antioxidants (e.g., ascorbicacid, sodium bisulfite, and the like); chelating agents (e.g.,ethylenediaminetetraacetic acid (EDTA) and the like); buffers (e.g.,acetates, citrates, phosphates, and the like); and/or agents for theadjustment of tonicity (e.g., sodium chloride, dextrose, and the like);or mixtures thereof.

Parenteral preparations can be enclosed in ampoules, disposablesyringes, or multiple dose vials made of glass, plastic, or othersuitable material. Buffers, preservatives, antioxidants, and the likecan be incorporated as required.

Where administered intravenously, suitable carriers includephysiological saline, phosphate buffered saline (PBS), and solutionscontaining thickening and solubilizing agents such as glucose,polyethylene glycol, polypropyleneglycol, and the like, and mixturesthereof. Liposomal suspensions including tissue-targeted liposomes mayalso be suitable as pharmaceutically acceptable carriers. These may beprepared according to methods known, for example, as described in U.S.Pat. No. 4,522,811.

The methods of treatment include delivery of the compounds of thepresent invention in a nano crystal dispersion formulation. Preparationof such formulations is described, for example, in U.S. Pat. No.5,145,684. Nano crystalline dispersions of HIV protease inhibitors andtheir method of use are described in U.S. Pat. No. 6,045,829. The nanocrystalline formulations typically afford greater bioavailability ofdrug compounds.

The methods of treatment include administration of the compoundsparenterally, for example, by IV, IM, SC, or depo-SC. When administeredparenterally, a therapeutically effective amount of about 0.2 mg/mL toabout 50 mg/mL is preferred. When a depot or IM formulation is used forinjection once a month or once every two weeks, the preferred doseshould be about 0.2 mg/mL to about 50 mg/mL.

The methods of treatment include administration of the compoundssublingually. When given sublingually, the compounds of the presentinvention should be given one to four times daily in the amountsdescribed above for IM administration.

The methods of treatment include administration of the compoundsintranasally. When given by this route, the appropriate dosage forms area nasal spray or dry powder, as is known to those skilled in the art.The dosage of the compounds of the present invention for intranasaladministration is the amount described above for IM administration.

The methods of treatment include administration of the compoundsintrathecally. When given by this route the appropriate dosage form canbe a parenteral dosage form as is known to those skilled in the art. Thedosage of the compounds of the present invention for intrathecaladministration is the amount described above for IM administration.

The methods of treatment include administration of the compoundstopically. When given by this route, the appropriate dosage form is acream, ointment, or patch. When topically administered, the dosage isfrom about 0.2 mg/day to about 200 mg/day. Because the amount that canbe delivered by a patch is limited, two or more patches may be used. Thenumber and size of the patch is not important. What is important is thata therapeutically effective amount of a compound of the presentinvention be delivered as is known to those skilled in the art. Thecompound can be administered rectally by suppository as is known tothose skilled in the art. When administered by suppository, thetherapeutically effective amount is from about 0.2 mg to about 500 mg.

The methods of treatment include administration of the compounds byimplants as is known to those skilled in the art. When administering acompound of the present invention by implant, the therapeuticallyeffective amount is the amount described above for depot administration.

Given a particular compound of the present invention and/or a desireddosage form and medium, one skilled in the art would know how to prepareand administer the appropriate dosage form and/or amount.

The methods of treatment include use of the compounds of the presentinvention, or acceptable pharmaceutical salts thereof, in combination,with each other or with other therapeutic agents, to treat or preventthe conditions listed above. Such agents or approaches includeacetylcholine esterase inhibitors such as tacrine(tetrahydroaminoacridine, marketed as COGNEX®), donepezil hydrochloride,(marketed as Aricept®) and rivastigmine (marketed as Exelon®),gamma-secretase inhibitors, anti-inflammatory agents such ascyclooxygenase II inhibitors, anti-oxidants such as Vitamin E orginkolides, immunological approaches, such as, for example, immunizationwith A-beta peptide or administration of anti-A-beta peptide antibodies,statins, and direct or indirect neurotropic agents such asCerebrolysin®, AIT-082 (Emilien, 2000, Arch. Neurol. 57:454), and otherneurotropic agents, and complexes with beta-secretase or fragmentsthereof.

Additionally, methods of treatment of the present invention also employthe compounds of the present invention with inhibitors of P-glycoprotein(P-gp). P-gp inhibitors and the use of such compounds are known to thoseskilled in the art. See, for example, Cancer Research, 53, 4595-4602(1993), Clin. Cancer Res., 2, 7-12 (1996), Cancer Research, 56,4171-4179 (1996), International Publications WO 99/64001 and WO01/10387. The blood level of the P-gp inhibitor should be such that itexerts its effect in inhibiting P-gp from decreasing brain blood levelsof the compounds of formula (I). To that end the P-gp inhibitor and thecompounds of formula (I) can be administered at the same time, by thesame or different route of administration, or at different times. Givena particular compound of formula (I), one skilled in the art would knowwhether a P-gp inhibitor is desirable for use in the method oftreatment, which P-gp inhibitor should be used, and how to prepare andadminister the appropriate dosage form and/or amount.

Suitable P-gp inhibitors include cyclosporin A, verapamil, tamoxifen,quinidine, Vitamin E-TGPS, ritonavir, megestrol acetate, progesterone,rapamycin, 10,11-methanodibenzosuberane, phenothiazines, acridinederivatives such as GF120918, FK506, VX-710, LY335979, PSC-833,GF-102,918, quinoline-3-carboxylic acid(2-{4-[2-(6,7-dimethyl-3,4-dihydro-1H-isoquinoline-2-yl)-ethyl]phenylcarbamoyl}-4,5-dimethylphenyl)-amide(Xenova), or other compounds. Compounds that have the same function andtherefore achieve the same outcome are also considered to be useful.

The P-gp inhibitors can be administered orally, parenterally, (via IV,IM, depo-IM, SQ, depo-SQ), topically, sublingually, rectally,intranasally, intrathecally, or by implant.

The therapeutically effective amount of the P-gp inhibitors is fromabout 0.1 mg/kg to about 300 mg/kg daily, preferably about 0.1 mg/kg toabout 150 mg/kg daily. It is understood that while a patient may bestarted on one dose, that dose may vary over time as the patient'scondition changes.

When administered orally, the P-gp inhibitors can be administered inusual dosage forms for oral administration as is known to those skilledin the art. These dosage forms include the usual solid unit dosage formsof tablets or capsules as well as liquid dosage forms such as solutions,suspensions or elixirs. When the solid dosage forms are used, it ispreferred that they be of the sustained release type so that the P-gpinhibitors need to be administered only once or twice daily. The oraldosage forms are administered to the patient one through four timesdaily. It is preferred that the P-gp inhibitors be administered eitherthree or fewer times a day, more preferably once or twice daily. Hence,it is preferred that the P-gp inhibitors be administered in solid dosageform and further it is preferred that the solid dosage form be asustained release form which permits once or twice daily dosing. It ispreferred that the dosage form used is designed to protect the P-gpinhibitors from the acidic environment of the stomach. Enteric coatedtablets are well known to those skilled in the art. In addition,capsules filled with small spheres each coated to protect from theacidic stomach, are also well known to those skilled in the art.

In addition, the P-gp inhibitors can be administered parenterally. Whenadministered parenterally they can be administered via IV, IM, depo-IM,SQ or depo-SQ.

The P-gp inhibitors can be given sublingually. When given sublingually,the P-gp inhibitors should be given one through four times daily in thesame amount as for IM administration.

The P-gp inhibitors can be given intranasally. When given by this routeof administration, the appropriate dosage forms are a nasal spray or drypowder as is known to those skilled in the art. The dosage of the P-gpinhibitors for intranasal administration is the same as for IMadministration.

The P-gp inhibitors can be given intrathecally. When given by this routeof administration the appropriate dosage form can be a parenteral dosageform as is known to those skilled in the art.

The P-gp inhibitors can be given topically. When given by this route ofadministration, the appropriate dosage form is a cream, ointment orpatch. Because of the amount of the P-gp inhibitors needed to beadministered the patch is preferred. However, the amount that can bedelivered by a patch is limited. Therefore, two or more patches may berequired. The number and size of the patch is not important, what isimportant is that a therapeutically effective amount of the P-gpinhibitors be delivered as is known to those skilled in the art.

The P-gp inhibitors can be administered rectally by suppository or byimplants, both of which are known to those skilled in the art.

It should be apparent to one skilled in the art that the exact dosageand frequency of administration will depend on the particular compoundsof the present invention administered, the particular condition beingtreated, the severity of the condition being treated, the age, weight,or general physical condition of the particular patient, or any othermedication the individual may be taking as is well known toadministering physicians who are skilled in this art.

Another embodiment of the present invention provides a method ofpreventing or treating at least one condition associated withamyloidosis using compounds with increased oral bioavailability(increased F values).

Another embodiment of the present invention provides methods forpreventing or treating at least one condition associated withamyloidosis, comprising administering to a host, a therapeuticallyeffective amount of at least one compound of formula (I), or at leastone pharmaceutically acceptable salt thereof, wherein R₁, R₂, and R_(C)are as previously defined, and wherein the compound has an F value of atleast 10%.

In another embodiment, the host is an animal.

In another embodiment, the host is human.

In another embodiment, the F value is greater than about 20%. In yet afurther embodiment, the F value is greater than about 30%.

Another embodiment of the present invention provides methods ofpreventing or treating at least one condition associated withamyloidosis using compounds with a high degree of selectivity.

Investigation of potential beta-secretase inhibitors produced compoundswith increased selectivity for beta-secretase over other aspartylproteases such as cathepsin D (catD), cathepsin E (catE), HumanImmunodeficiency Viral (HIV) protease, and renin. Selectivity wascalculated as a ratio of inhibition (IC₅₀) values in which theinhibition of beta-secretase was compared to the inhibition of otheraspartyl proteases. A compound is selective when the IC₅₀ value (i.e.,concentration required for 50% inhibition) of a desired target (e.g.,beta-secretase) is less than the IC₅₀ value of a secondary target (e.g.,catD).

Alternatively, a compound is selective when its binding affinity isgreater for its desired target (e.g., beta-secretase) versus a secondarytarget (e.g., catD).

Accordingly, methods of treatment include administering selectivecompounds of formula (I) having a lower IC₅₀ value for inhibitingbeta-secretase, or greater binding affinity for beta-secretase, than forother aspartyl proteases such as catD, catE, HIV protease, or renin. Aselective compound is also capable of producing a higher ratio ofdesired effects to adverse effects, resulting in a safer method oftreatment.

Exemplary compounds of formula (I) are provided in the Examples below.

EXAMPLE 1 Exemplary Formula (I) Compounds

EX- AMPLE No. Compound 1-1.

4-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-2.

4-[1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-3.

4-[1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-4.

4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclo-hexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-5.

4-[4-(2-Amino-ethoxyimino)-1-(3-tert-butyl-phenyl)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-6.

4-[5-(3-tert-Butyl-phenyl)-2-hydroxyimino-hexahydro-pyrimidin-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-7.

4-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-8.

4-[1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-9.

4-[1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-10.

4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-11.

4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-benzo[c]isoxazol-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-12.

4-[1-(3-tert-Butyl-phenyl)-4-methylcarbamoylmethylene-cyclo-hexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-13.

{4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-methylcarbamoyl-butylamino]-cyclohexylidene}-aceticacidmethyl ester 1-14.

4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethylidene)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide1-15.

4-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-16.

4-[1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-17.

4-[1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-18.

4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclo-hexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1.19.

4-[4-(2-Amino-ethoxyimino)-1-(3-tert-butyl-phenyl)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-20.

4-[5-(3-tert-Butyl-phenyl)-2-hydroxyimino-hexahydro-pyrimidin-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-21.

4-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-22.

4-[1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-23.

4-[1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-24.

4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-25.

4-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-benzo[c]isoxazol-5-ylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-26.

4-[1-(3-tert-Butyl-phenyl)-4-methylcarbamoylmethylene-cyclo-hexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-27.

{4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-phenylcarbamoyl-butylamino]-cyclohexylidene}-aceticacidmethyl ester 1-28.

4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethylidene)-cyclohexyl-amino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide1-29.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-(1H-imidazol-2-yl)-butylamino]-cyclohexanoneoxime 1-30.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-butylamino]-cyclohexanoneoxime 1-31.

4-(3-tert-Butyl-phenyl)-4-[3-(3,5-difluoro-phenoxy)-2-hydroxy-propylamino]-cyclohexanoneoxime 1-32.

4-(3-tert-Butyl-phenyl)-4-[3-(3,5-difluoro-benzenesulfonyl)-2-hydroxy-propylamino]-cyclohexanoneoxime 1-33.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-4-oxo-butylamino]-cyclohexanoneoxime 1-34.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-pentylamino]-cyclohexanoneoxime 1-35.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-pentylamino]-cyclohexanoneoxime 1-36.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-tetrazol-1-yl-butylamino]-cyclohexanoneoxime 1-37.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-(5-trifluoromethyl-[1,3,4]oxadiazol-2-yl)-butylamino]-cyclohexanoneoxime 1-38.

4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-([1,2,4]thiadiazol-5-ylamino)-butylamino]-cyclohexanoneoxime 1-39.

3-[3-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propylamino]-4-methylamino-cyclobut-3-ene-1,2-dione1-40.

3-[3-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propylamino]-4-methoxy-cyclobut-3-ene-1,2-dione1-41.

4-(3-tert-Butyl-phenyl)-4-[2-hydroxy-4-(3-propyl-thiophen-2-yl)-3-([1,2,4]thiadiazol-5-ylamino)-butylamino]-cyclohexanoneoxime1-42.

1-(5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino)-4-(3,5-difluorophenyl)butan-2-ol1-43.

1-(5-(3-tert-butylphenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamino)-4-(3,5-difluorophenyl)butan-2-ol

EXPERIMENTAL PROCEDURES

The compounds and the methods of treatment of the present invention canbe prepared by one skilled in the art based on knowledge of thecompound's chemical structure. The chemistry for the preparation of thecompounds employed in the methods of treatment of this invention isknown to those skilled in the art. In fact, there is more than oneprocess to prepare the compounds employed in the methods of treatment ofthe present invention. Specific examples of methods of preparation canbe found in the art. For examples, see Zuccarello et al., J. Org. Chem.1998, 63, 4898-4906; Benedetti et al., J. Org. Chem. 1997, 62,9348-9353; Kang et al., J. Org. Chem. 1996, 61, 5528-5531; Kempf et al.,J. Med. Chem. 1993, 36, 320-330; Lee et al., J. Am. Chem. Soc. 1999,121, 1145-1155; and references cited therein; Chem. Pharm. Bull. (2000),48(11), 1702-1710; J. Am. Chem. Soc. (1974), 96(8), 2463-72; Ind. J.Chem., §B: Organic Chemistry Including Medicinal Chemistry (2003),42B(4), 910-915; and J. Chem. Soc. §C: Organic (1971), (9), 1658-10. Seealso U.S. Pat. Nos. 6,150,530, 5,892,052, 5,696,270, and 5,362,912, andreferences cited therein, which are incorporated herein by reference.

¹H and ¹³C NMR spectra were obtained on a Varian 400 MHz, Varian 300MHz, or Bruker 300 MHz instrument and as described in the aboveexamples. Unless otherwise stated, HPLC samples were analyzed using aYMC ODS-AQ S-3 120 A 3.0×50 mm cartridge, with a standard gradient from5% acetonitrile containing 0.01% heptafluorobutyric acid (HFBA) and 1%isopropanol in water containing 0.01% HFBA to 95% acetonitrilecontaining 0.01% HFBA and 1% isopropanol in water containing 0.01% HFBAover 5 minutes. Mass spec samples were performed with electron sprayionization (ESI).

Exemplary HPLC Procedures

Various High Pressure Liquid Chromatography (HPLC) procedures employedthe following methods:

Method [1] utilizes a 20% [B]: 80% [A] to 70% [B]: 30% [A] gradient in1.75 min, then hold, at 2 mL/min, where [A]=0.1% trifluoroacetic acid inwater; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [2] utilizes a 50% [B]: 50% [A] to 95% [B]: 5% [A] gradient in2.5 min, then hold, at 2 mL/min, where [A]=0.1% trifluoroacetic acid inwater; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [3] utilizes a 5% [B]: 95% [A] to 20% [B]: 80% [A] gradient in2.5 min, then hold, at 2 mL/min, where [A]=0.1% trifluoroacetic acid inwater; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [4] utilizes a 20% [B]: 80% [A] to 70% [B]: 30% [A] gradient in2.33 min, then hold, at 1.5 mL/min, where [A]=0.1% trifluoroacetic acidin water; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [5] utilizes a 50% [B]: 50% [A] to 95% [B]: 5% [A] gradient in3.33 min, then hold, at 1.5 mL/min, where [A]=0.1% trifluoroacetic acidin water; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [6] utilizes a 5% [B]: 95% [A] to 20% [B]: 80% [A] gradient in3.33 min, then hold, at 1.5 mL/min, where [A]=0.1% trifluoroacetic acidin water; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [7] utilizes a 20% [B]: 80% [A] to 70% [B]: 30% [A] gradient in1.75 min, then hold, at 2 mL/min, where [A]=0.1% trifluoroacetic acid inwater; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×30 cm column, 3 micron packing, 210 nm detection, at35° C.

Method [8] utilizes a YMC ODS-AQ S-3 120 A 3.0×50 mm cartridge, with astandard gradient from 5% acetonitrile containing 0.01%heptafluorobutyric acid (HFBA) and 1% isopropanol in water containing0.01% HFBA to 95% acetonitrile containing 0.01% HFBA and 1% isopropanolin water containing 0.01% HFBA over 5 min.

Method [9] utilizes a 20% [B]: 80% [A] to 70% [B]: 30% [A] gradient in10.0 min, then hold, at 1.5 mL/min, where [A]=0.1% trifluoroacetic acidin water; [B]=0.1% trifluoroacetic acid in acetonitrile on a PhenomenexLuna C18 (2) 4.6 mm×3 cm column, 3 micron packing, 210 nm detection, at35° C.

EXAMPLE 2 General Preparation of Formula (I) Compounds

The general synthesis of compounds of formula (I) are shown in the aboveScheme 1, and Schemes 2 and 3 below. In Scheme 1, chiral epoxides (1),which were derived from amino acids and are known in the art (see Luly,J. R. et al. J. Org. Chem. 1987, 52, 1487; Tucker, T. J. et al. J. Med.Chem. 1992, 35, 2525), were treated with 1.5-5 equivalents of primaryamine H₂N—R_(C) in a C₁-C₆ alcoholic solvent, such as ethanol,isopropanol, or sec-butanol to effect ring opening of the epoxide. Thereactions can be run at temperatures ranging from about 20-25° C. up toabout the reflux temperature of the alcohol employed. The preferredtemperature range for conducting the reaction is between 40° C. and therefluxing temperature of the alcohol employed. A more preferredembodiment is to perform this reaction at reflux in isopropanol.

The resulting amino alcohol is protected with capping group P₂.Appropriate protecting groups such as tert-butoxycarbonyl (Boc) orbenzyloxycarbonyl (Cbz) may be introduced via treatment with theappropriate anhydride or carbamoyl chloride as known in the art in orderto provide compounds of type 2. It is preferred to select protectinggroups P₂ which may be orthogonally removed independently from P₁.

The protecting group P₁ is removed affording the corresponding amine bymeans known to those skilled in the art for removal of amine protectinggroups. For example, it is preferred to remove the preferred protectinggroup, Boc, by dissolving 2 in a trifluoroacetic acid/dichloromethane(1/1) mixture. When complete, the solvents are removed under reducedpressure yielding the corresponding amine (3) (as the correspondingsalt, i.e. trifluoroacetic acid salt) which is used without furtherpurification. If desired, the amine can be purified further by meanswell known to those skilled in the art, such as, for example,recrystallization. Further, if the non-salt form is desired, it also canbe obtained by means known to those skilled in the art, such as, forexample, preparing the free base amine via treatment of the salt withmild basic conditions. Additional Boc deprotection conditions anddeprotection conditions for other protecting groups can be found in T.W. Green and P. G. M. Wuts in Protecting Groups in Organic Chemistry,3^(rd) edition, John Wiley and Sons, 1999.

The addition of the group R may be achieved by a variety of methodsknown in the art, depending on the nature of R, and can be found in R.C. Larock's Comprehensive Organic Transformations, VCH Publishers, 1989,e.g., pp. 972, 979, and 981. Removal of the protecting group P₂ bymethods known in the art would then afford 4.

EXAMPLE 3 Alternative Preparation of Formula (I) Compounds

An alternative approach was to use a common advanced intermediate 7 bywhich a reactive group could be converted to yield compounds 4. Epoxides1 were treated with 1.5-5 equivalents of primary amine H₂N—R_(c1) in analcoholic solvent, such as ethanol, isopropanol, or sec-butanol toeffect ring opening of the epoxide. In an embodiment, this reaction isprepared at elevated temperatures from 40° C. to reflux. In anotherembodiment, this reaction is performed at reflux in isopropanol. Theresulting amino alcohol 5 was then deprotected.

When R_(c1) contains a labile functional group, such as an aryl iodide,aryl bromide, aryl trifluoromethanesulfonate, or aryl boronic ester,which may be converted into R_(C) via transition metal-mediatedcoupling, this allows for the rapid synthesis of a variety of analogs 4.Such conversions may include Suzuki (aryl boronic acid or boronic esterand aryl halide), Negishi (arylzinc and aryl or vinyl halide), andSonogashira (arylzinc and alkynyl halide) couplings. Subsequent to thecoupling reaction, the protecting group P₂ is removed in methods knownin the art to yield compounds 4.

EXAMPLE 4 Alternative Preparation of Formula (I) Compounds

As described herein, one embodiment of the present invention providesfor compounds of formula 8 as shown above in Scheme 3. These compoundsmay be made by methods known to those skilled in the art from startingcompounds that are also known to those skilled in the art. The processchemistry is further well known to those skilled in the art. A suitableprocess for the preparation of compounds of formula 8 is set forth inScheme 3 above.

EXAMPLE 5 Prepatation of8-(3-ISOPROPYLPHENYL)-1,4-DIOXA-SPIRO[4.5]DECANE-8-AMINE ACETATE (11)

Step 1. Preparation of8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-alcohol (9)

A solution of 3-bromoisopropylbenzene (25 mmol) in 20 mL of dry THF wasadded dropwise over 20 min to 1.22 g (50 mmol) of magnesium turnings in10 mL of refluxing THF under nitrogen and the mixture was refluxed foran additional 25 min to form the Grignard reagent. The Grignard solutionwas cooled and added by cannula to a suspension of CuBr-dimethylsulfidecomplex (0.52 g, 2.5 mmol) in dry THF at −25° C. The suspension wasstirred at −25° C. for 20 min, and then a solution of 1,4cyclohexanedione, monoethylene ketal (3.9 g, 25 mmol) in 15 mL of THFwas added dropwise over 5 min. The mixture was allowed to gradually warmto ambient temperature. After chromatography over silica gel, elutingwith 20% to 30% ethyl acetate in heptane, alcohol 9 (5.6 g, 20 mmol,80%) was isolated as a colorless oil which crystallized to a white solidon cooling: ¹H NMR (CDCl₃) δ 7.39 (s, 1 H), 7.33 (m, 1H), 7.28 (t, J=7.5Hz, 1H), 7.13 (d, J=7.5 Hz, 1H), 4.0 (m, 4H), 2.91 (hept, J=7 Hz, 1H),2.15 (m, 4H), 1.82 (br d, J=11.5 Hz, 2H), 1.70 (brd, J=11.5 Hz, 2H),1.25 (d, J=7 Hz, 6 H); MS (CI) m/z 259.2 (M−OH).

Step 2. Preparation of8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-azide (10)

8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-alcohol 9 (5.5 g, 20mmol) was reacted with sodium azide (2.9 g, 45 mmol) and trifluoroaceticacid (TFA, 13 mL, 170 mmol) in 120 mL of CH₂Cl₂ at 0° C., allowing thereaction to stir 2 h after dropwise addition of the TFA. The reactionwas quenched by dropwise addition of 18 mL of concentrated NH₄OH.

The mixture was taken up in water, ethyl acetate, and heptane, and theorganic phase was washed three more times with water and once withbrine. The solution was dried (Na₂SO₄), filtered, concentrated, andchromatographed over silica gel, eluting with 3% acetone in heptane.Concentration of the product-containing fractions afforded 2.2 g (7.3mmol, 36%) of 10 as a colorless oil: ¹H NMR (CDCl₃) δ 7.33-7.26 (m, 3H), 7.17 (m, 1 H), 3.98 (m, 4 H), 2.92 (hept, J=7 Hz, 1 H), 2.2-2.12 (m,2 H), 2.07-1.95 (m, 4 H), 1.72 (m, 2 H), 1.26 (d, J=7 Hz, 6 H).

Step 3. Preparation of8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amine acetate (11)

2.2 g (7.3 mmol) of8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-azide (10) in 200 mLof ethanol was reduced under 16 psi of hydrogen in the presence of 0.7 gof 10% palladium on carbon for 4.5 h. Filtration and removal of solventswith a toluene azeotrope affords a white solid which was triturated withpentane to yield 2.14 g (6.4 mmol, 87%) of 11 as a white solid: ¹H NMR(CDCl₃) δ 7.37-7.33 (m, 2 H), 7.30-7.26 (m, 1 H), 7.13 (d, J=7.5 Hz, 1H), 5.91 (br, 3 H), 3.96 (m, 4 H), 2.90 (hept., J=7 Hz, 1 H), 2.32 (m, 2H), 2.03 (s, 3 H), 2.0-1.85 (m, 4 H), 1.63 (m, 2 H), 1.25 (d, J=7 Hz, 6H); MS (CI) m/z 259.2 (M-NH₂).

EXAMPLE 6 Exemplary Preparation of Oxime Precursor

EXAMPLE 7 Exemplary Prepatation of an Oxime

EXAMPLE 8 Exemplary Preparation of a Hydrazone

EXAMPLE 9 Preparation ofN-((1S,2R)-1-(3,5-DIFLUOROBENZYL)-2-HYDROXY-3-{[1-(3-ISOPROPYLPHENYL)CYCLOHEXAN-4-ONE]AMINO}PROPYL)ACETAMIDE (15)

Step 1. Preparation of tert-butyl(1S,2R)-1-(3,5-difluorobenzyl)-2-hydroxy-3-{8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amino}propylcarbamate(13)

8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amine acetate 11 (3.2mmol) was neutralized and reacted with[2-(3,5-Difluoro-phenyl)-1-oxiranyl-ethyl]-carbamic acid tert-butylester 12 (0.6 g, 2.0 mmol) in refluxing isopropanol (15 mL) for 15.5 h.The reaction mixture was concentrated and chromatographed over silicagel, eluting with 4% methanol (containing 2% of NH₄OH) in CH₂Cl₂ toseparate the crude product from excess8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amine. The crudeproduct was then re-chromatographed over silica gel, eluting with 10% to20% acetone in CH₂Cl₂ yielding 0.600 g (1.04 mmol, 52%) of 13 as acolorless oil: ¹H NMR (CDCl₃) δ 7.27-7.20 (m, 3 H), 7.09 (d, J=7 Hz, 1H), 6.69 (m, 2 H), 6.63 (m, 1 H), 4.64 (d, J=9 Hz, 1H), 3.95 (m, 4H),3.72 (m, 1H), 3.28 (m, 1H), 2.88 (m, 2H), 2.69 (dd, J=8.5, 14 Hz, 1 H),2.32 (m, 2 H), 2.15 (m, 2 H), 1.99-1.86 (m, 4 H), 1.63 (m, 2 H), 1.35(s, 9 H), 1.24 (d, J=7 Hz, 6 H); MS (CI) m/z 575.4 (MH+)

Step 2. Preparation ofN-((1S,2R)-1-(3,5-difluorobenzyl)-2-hydroxy-3-{8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amino}propyl)acetamide(14)

Tert-butyl(1S,2R)-1-(3,5-difluorobenzyl)-2-hydroxy-3-{8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amino}propylcarbamate13 (0.600 g, 1.04 mmol) was deprotected, acetylated, and saponifiedyielding, after chromatography on silica gel, eluting with 32.5% acetoneand 2.5% methanol in CH₂Cl₂, acetamide 14 (335 mg, 0.65 mmol, 62%) as awhite solid by concentration from ethyl ether: ¹H NMR (CDCl₃) δ7.31-7.26 (m, 3 H), 7.15 (m, 1 H), 6.69-6.61 (m, 3 H), 5.9 (br, 1 H),4.13 (m, 1 H), 3.95 (m, 4 H), 3.48 (m, 1 H), 2.92-2.83 (m, 2 H), 2.73(dd, J=8.5, 14 Hz, 1 H), 2.45-2.25 (m, 4 H), 2.10 (m, 2 H), 1.88 (s+m, 5H), 1.62 (m, 2 H), 1.25 (d, J=7 Hz, 6 H); MS (CI) m/z 517.4 (MH+).

Step 3. Preparation ofN-((1S,2R)-1-(3,5-difluorobenzyl)-2-hydroxy-3-{[1-(3-isopropylphenyl)cyclohexan-4-one]amino}propyl)acetamide(15)

ToN-((1S,2R)-1-(3,5-difluorobenzyl)-2-hydroxy-3-{8-(3-isopropylphenyl)-1,4-dioxa-spiro[4.5]decane-8-amino}propyl)acetamide14 (255 mg, 0.49 mmol) in 5 mL of ethanol and 5 mL of water was added 6mL of trifluoroacetic acid, and the mixture was refluxed for 2 h undernitrogen. It was concentrated and taken up in aqueous 10% Na₂CO₃ andethyl acetate. The organic phase was washed twice more with 10% Na₂CO₃and then with brine. It was dried over Na₂SO₄, and concentrated to acolorless oil. Evaporation in vacuo from ethyl ether affords 15 (140 mg,0.30 mmol, 60%) as a white solid: ¹H NMR (CDCl₃) δ 7.35-7.18 (m, 4 H),6.71-6.64 (m, 3 H), 5.65 (br, 1 H), 4.12 (m, 1 H), 3.43 (m, 1 H),2.95-2.90 (m, 2 H), 2.75 (dd, J=8.5, 14 Hz, 1 H), 2.64 (m, 2 H),2.4-2.25 (m, 8 H), 1.87 (s, 3 H), 1.25 (d, J=7 Hz, 6 H); MS (CI) m/z473.4 (MH+). The LC-MS spectrum in methanol solvent showed a smallsignal at 505.4 (MH+CH₃OH) due to hemiketal formation. IR (diffusereflectance) 3311, 2958, 1710, 1646, 1628, 1595, 1550, 1544, 1460, 1372,1315, 1116, 983, 846, 707 cm⁻¹.

MS (EI) m/z (relative intensity) 472 (M+, 6), 472 (6), 417 (5), 416(33), 415 (99), 398 (8), 397 (30), 327 (11), 244 (9), 215 (13), 214 (6).HRMS (ESI) calculated for C₂₇H₃₄N₂O₃F₂+H₁ 473.2615, found 473.2627.Anal. Calc'd for C₂₇H₃₄F₂N₂O₃+0.5H₂O: C, 67.34; H, 7.33; N, 5.82. Found(av): C, 67.89; H, 7.32; N, 5.86.

EXAMPLE 10 Preparation ofN-[3-[1-(3-TERT)-BUTYL-PHENYL)-4-HYDROXYIMINO-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE

To a solution ofN-[3-[1-(3-tert)-Butyl-phenyl)-4-oxo-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide(0.208 g, 0.43 mmol) in ethanol (4 mL) was added hydroxylaminehydrochloride (0.074 g, 1.07 mmol) and sodium acetate (0.17 g, 2.05mmol). The reaction mixture was stirred at room temperature for 2.5 hprior to partitioning between H₂O and CH₂Cl₂. The organic layer wasseparated, dried (Na₂SO₄) and concentrated under reduced pressure. Theresidue was purified by flash chromatography (5% MeOH/CH₂Cl₂) to yieldthe desired product (0.11 g, 53%). MS (ESI): 502.2 (M+H). See Bravo, P.,et al., J. Fluorine Chem., 59 (1992), 153-56.

EXAMPLE 11 Preparation ofN-[3-[1-(3-TERT)-BUTYL-PHENYL)-4-METHOXYIMINO-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE

N-[3-[1-(3-tert)-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamidewas prepared according to essentially the same procedure as described inEXAMPLE 10. MS (ESI): 516.3 (M+H).

EXAMPLE 12N-[3-[1-(3-TERT)-BUTYL-PHENYL)-4-ETHOXYIMINO-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE

N-[3-[1-(3-tert)-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamidewas prepared according to essentially the same procedure as described inEXAMPLE 10. MS (ESI): 530.2 (M+H).

EXAMPLE 13 2-AMINOETHANOL HYDROCHLORIDE

Tert-Butyl N-hydroxycarbamate (Aldrich, 2.64 g, 19.8 mmol) was dissolvedin 1,8-diazabicyclo[5.4.0]undec-7-ene (3.0 mL, 20 mmol) and2-bromoethanol (1.7 mL, 24 mmol). The reaction mixture was allowed tostir at room temperature overnight and quenched with 1N HCl. The productwas extracted with CH₂Cl₂, dried (Na₂SO₄), and concentrated underreduced pressure. The residue was purified by flash chromatography(Hexane:EtOAc, 1:1) yielding the desired product (2.48 g, 71%). MS(ESI): 200.1 (M+Na).

The N-Boc intermediate was deprotected by treatment with 4 N HCl indioxane. The reaction mixture was allowed to stir at room temperaturefor 2 h prior to concentration under reduced pressure. See Jones, D. S.,et al., Tet. Lett., 41, (2000) 1531-33.

EXAMPLE 14N-[3-[1-(3-TERT)-BUTYL-PHENYL)-4-(2-HYDROXY-ETHOXYIMINO-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE

N-[3-[1-(3-tert)-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamidewas prepared according to essentially the same procedure as described inEXAMPLE 10. MS (ESI): 546.3 (M+H). See Jones, D. S., et al., Tet. Lett.,41, (2000) 1531-33.

EXAMPLE 15 Preparation ofN-{1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-3-[4-METHOXYIMINO-1-(3-R—PHENYL)-CYCLOHEXYLAMINO]-PROPYL}-ACETAMIDE

25 mg (0.04 mmol) of theN-[3-[1-(3-Bromo-phenyl)-4-oxo-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide15 was dissolved in 1 mL DME and placed in a 4 mL reaction vial. UnderN₂(g), a solution of the boronic acid (0.06 mmol),tetrakis(triphenylphosphine) palladium(0) (0.006 mmol), and 0.125 mL ofaqueous 2M Na₂CO₃ dissolved in 1 mL DME was added to the reactionmixture. The reaction was then stirred at 95° C. for 15 h. The reactionmixture was then concentrated yielding product 16.

The product 16 (0.048 mmol) was then dissolved in 1 mL ethanol andplaced in a 4 mL reaction vial. Methoxylamine hydrochloride (0.23 mmol)and sodium acetate (0.13 mmol) are added in the vial. The reaction wasthen stirred for 2.5 h at room temperature. The reaction mixture wasthen concentrated and the product 17 was isolated via preparative HPLC:Method [1].

EXAMPLE 16 Procedure ofN-[3-[1-(3-BROMO-PHENYL)-4-OXO-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE(19)

Step 1: Procedure of 2-Methyl-propane-2-sulfinic acid(1,4-dioxa-spiro[4.5]dec-8-ylidene)-amide (15)

An oven dried round-bottom flask was cooled to room temperature byflushing with N₂(g) for 30 min. 1,4-Dioxa-spiro[4.5]decan-8-one (1.35 g,8.66 mmol) (dissolved in 12 mL THF), 2-Methyl-propane-2-sulfinic acidamide (1.0 g, 8.25 mmol) (dissolved in THF), and titanium(IV) ethoxide(3.77 g, 16.50 mmol) were added. The reaction was stirred for 4 h atroom temperature. To the mixture was added 15 mL saturated NaHCO₃followed by filtration and an EtOAc rinse. The organic layer was driedwith MgSO₄, filtered and concentrated under reduced pressure yielding0.98 g of Compound 15.

MS m/z 260.1; retention time: 0.754, method [8].

Step 2: Procedure for8-(3-Bromo-phenyl)-1,4-dioxa-spiro[4.5]dec-8-ylamine Hydrochloride (16)

Two oven dried round-bottom flask were cooled to room temperature byflushing with N₂(g). n-Butyl Lithium (2.5 M in hexanes) (0.46 g, 7.14mmol) was added dropwise to a stirring solution of1-Bromo-3-iodo-benzene (2.02 g, 7.14 mmol) in 3.2 mL toluene at 0° C.The reaction stirred from 0° C. to room temperature over 2 h. A separatesolution of compound 15 (0.98 g, 3.4 mmol) and AlMe₃ (0.269 g, 3.74mmol) were added to a second flask cooled to −78° C. and stirred for 10min. This second mixture was added by cannula to the first. The combinedmaterial was at 0° C. and allowed to reach room temperature over 3 h.The reaction was then quenched with Na₂SO₄.6H₂O. MgSO₄ was added to thereaction mixture, which was then filtered and concentrated under reducedpressure. The reaction provided 1.6 g of crude material. A column onsilica gel (50% EtOAc:hexanes) provided 0.29 g of pure material. Thepure material was treated with 0.69 mL 4M HCl in dioxanes and stirredfor 1 h at room temperature. The reaction mixture was then placed underreduced pressure. 0.23 g of Compound 16 were recovered.

MS m/z 295.0 (M-NH₂); retention time: 0.979, method [8].

Step 3: Procedure for[3-[8-(3-Bromo-phenyl)-1,4-dioxa-spiro[4.5]dec-8-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-carbamicacid tert-butyl ester (17)

Compound 16 was dissolved in 1 mL MeOH and added to a round bottomflask. 2M NaOH was added until the pH was approximately 10. The reactionmixture was rinsed six times with CH₂Cl₂. The organic layer was driedwith MgSO₄, filtered and concentrated under reduced pressure to get 0.16grams of product. The product was then dissolved in 1.0 mL isopropylalcohol and added to a sealed tube containing[2-(3,5-Difluoro-phenyl)-1-oxiranyl-ethyl]-carbamic acid tert-butylester (0.72 mmol). The reaction was heated to 80° C. over night. Thereaction was concentrated by reduced pressure yielding Compound 17.

MS m/z 611.1; retention time: 1.919, method [7].

Step 4: Procedure forN-[3-[8-(3-Bromo-phenyl)-1,4-dioxa-spiro[4.5]dec-8-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide(18)

Compound 17 was dissolved in 1 mL (1:1) trifluoroacetic acid (TFA) andCH₂Cl₂. The reaction stirred at room temperature for 2 h andconcentrated under reduced pressure. The residue was dissolved in 4 mLCH₂Cl₂ and N-Methylmorpholine (NMM) (3.12 mmol). The reaction wasstirred at 0° C. Acetic Acid (0.76 mmol) was added slowly to thereaction mixture and the mixture stirred at 0° C. for five min. Then1-Hydroxylbenzotriazole hydrate (HOBt) (0.76 mmol) and1-Ethyl-3-(3′-Dimethylaminopropyl)carbodiimide Hydrochloride (EDC.HCl)(0.76 mmol) were added sequentially. The reaction was stirred at roomtemperature for two h. CH₂Cl₂ was removed by reduced pressure and theresidue dissolved in EtOAc. The organic layer was rinsed with asaturated NaHCO₃ solution three times and once with Brine. The organiclayer was dried with MgSO₄, filtered and concentrated under reducedpressure. Compound 22 was purified by preparative HPLC.

MS m/z 509.0; retention time: 1.335, method [7].

Step 5: Procedure forN-[3-[1-(3-Bromo-phenyl)-4-oxo-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide(19)

Compound 18 (0.4 g, 0.78 mmol), p-Toluenesulfonic acid monohydrate(TsOH) (0.16 g, 0.84 mmol), and poly(Ethylene glycol) (8.9 g, 143.4mmol) were added to 25 mL benzene. The reaction was heated to 100° C.for 30 min. The benzene was removed under reduced pressure and freshbenzene was added. The resulting mixture was treated with saturatedNaHCO₃ and extracted CH₂Cl₂. The organic layer was washed with brine anddried with MgSO₄, filtered and concentrated under reduced pressureproviding 0.4 g of Compound 19.

MS m/z 553.1; retention time: 1.523, method [7].

EXAMPLE 17N-{1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-3-[8-(3-PYRAZOL-1-YL-PHENYL)-1,4-DIOXA-SPIRO[4.5]DEC-8-YLAMINO]-PROPYL}-ACETAMIDE(21)

Compound 18 (0.4 g, 0.72 mmol), pyrazole (0.059 g, 0.87 mmol), andcesium carbonate (0.47 g, 1.45 mmol) were added to a round-bottom flask.Diglyme was added to trans-1,2-diaminocyclohexane (0.0082 g, 0.072mmol). This mixture was added to Copper(I) Iodide (0.014 g, 0.072 mmol).The mixture was then added to the round-bottom flask. The reactionmixture was then heated to 130° C. for 4 days. The crude material waspurified by preparative HPLC (13.0 mg) yielding Compound 20.

¹H NMR (CD₃OD) δ 7.87 (s, 1H), 7.72-7.65 (m, 2H), 7.52-7.46 (m, 1H),6.82-6.79 (m, 3H), 4.00-3.87 (m, 4H), 3.57-3.54 (m, 1H), 3.23-3.17 (m,1H), 2.83-2.65 (m, 3H), 2.58-2.53 (m, 1H), 2.27-2.19 (m, 2H), 1.87 (s,2H), 1.80 (s, 2H), 1.80 (s, 3H), 1.51-1.29 (m, 4H).

MS m/z 541.2; retention time: 1.412, method [7].

EXAMPLE 18N-{1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-3-[4-METHOXYIMINO-1-(3-PYRAZOL-1-YL-PHENYL)-CYCLOHEXYLAMINO]-PROPYL}-ACETAMIDE(21)

Compound 20 was treated with a (1:1) 2M HCl and THF solution (10 mL) andrefluxed overnight. Almost all the THF was removed under reducedpressure and 10% NaOH was added until the pH was approximately 10. Thereaction mixture was then rinsed six times with CH₂Cl₂. The organiclayer was rinsed with MgSO₄, filtered and concentrated under reducedpressure to provide 0.044 g of about 85% pure product. The ketone wasthen transferred to a round-bottom flask containing CH₃ONH₂.HCl (0.013g, 0.20 mmol), NaOAc (0.032 g, 0.39 mmol), and 5 mL EtOH. The reactionstirred at room temperature for 2.5 h. The reaction was quenched withH₂O and extracted with CH₂Cl₂. The CH₂Cl₂ was removed under reducedpressure and the crude material was purified by preparative HPLC (9.0mg) yielding Compound 21.

¹H NMR (CD₃OD) δ 8.35-8.34 (bs, 1H), 8.12 (s, 1H), 7.91-7.88 (d, J=9 Hz,1H), 7.77 (s, 1H), 7.72-7.63 (m, 2H), 6.79-6.73 (m, 3H), 6.58-6.57 (m,1H), 3.86-3.83 (m, 1H), 3.77 (s, 3H), 3.58-3.54 (m, 1H), 3.23-3.15 (m,2H), 2.93-2.89 (m, 2H), 2.76-2.73 (bs, 2H), 2.57-2.48 (m, 2H), 2.21-2.17(m, 2H), 2.13-2.03 (m, 2H), 1.71 (s, 3H).

MS m/z 526.2; retention time: 1.464, method [7].

EXAMPLE 19N-[3-[1-(3-TERT-BUTYL-PHENYL)-3-METHYLENE-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDEStep 1

3-oxo-cyclohexanecarboxylic acid (2.00 g, 14.1 mmol),2-trimethylsilylethanol (2.5 mL, 17.4 mmol), 4-dimethylaminopyridine(148 mg, 1.21 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (3.44 g, 17.9 mmol) in methylene chloride (14 mL) wasstirred for 18 h. The solution was diluted with 10% aqueous hydrochloricacid and extracted with methylene chloride. The combined organicextracts were dried over magnesium sulfate, filtered, and concentratedto yield 3.41 g (100% yield) of 3-oxo-cyclohexanecarboxylic acid2-trimethylsilanyl-ethyl ester as a clear oil.

¹H NMR (300 MHz, CDCl₃) δ 4.16 (m, 2H), 2.75 (m, 1H), 2.55 (d, J=7.9 Hz,2H), 2.36 (m, 2H), 2.08 (m, 2H), 1.82 (m, 2H), 0.98 (m, 2H), 0.04 (s,9H).

Step 2

A solution of 1.6 M n-butyllithium in hexanes (14.0 mL, 22.4 mmol) wasadded to a heterogeneous mixture of methyltriphenylphosphonium bromide(8.02 g, 22.4 mmol) in tetrahydrofuran (50 mL) at −10° C. After stirringfor 30 min at −10° C., the yellow slurry was cooled to −78° C. and3-oxo-cyclohexanecarboxylic acid 2-trimethylsilanyl-ethyl ester (3.41mg, 14.1 mmol) in tetrahydrofuran (20 mL) was added. After stirring for10 min at −78° C., the dry ice/acetone bath was removed and theheterogeneous mixture was stirred for 3 h, during which time thesolution warmed to ambient temperature. The heterogeneous mixture wasconcentrated and the residue was flash chromatographed with 99:1, 49:1,24:1, and 23:2 hexanes:etheyl acetate as the eluant to yield 3.38 g(100% yield) of 3-methylene-cyclohexanecarboxylic acid2-trimethylsilanyl-ethyl ester as a clear oil.

¹H NMR (300 MHz, CDCl₃) δ 4.68 (s, 2H), 4.16 (m, 2H), 2.51 (m, 1H), 2.24(broad m, 3H), 1.98 (m, 2H), 1.86 (m, 1H), 1.55 (m, 1H), 1.38 (m, 1H),0.98 (m, 2H), 0.05 (s, 9H).

Step 3

A 1.6 M solution of n-butyllithium (12.0 mL, 19.2 mmol) was added to asolution of dicyclohexylamine (3.7 mL, 18.6 mmol) in toluene (40 mL).After stirring for 5 min, 3-methylene-cyclohexanecarboxylic acid2-trimethylsilanyl-ethyl ester (3.45 g, 14.4 mmol) was added. Afterstirring for 30 min, 1-bromo-3-tert-butyl-benzene (3.16 g, 14.8 mmol)was added followed by the simultaneous addition oftri-tert-butylphosphonium tetrafluoroborate (220 mg, 758 mmol) andtris(dibenzylideneacetone)dipalladium(0)-chloroform adduct (380 mg, 367mmol). The solution was placed into a preheated oil bath at 60° C. Afterstirring for 16 h, the solution was directly flash chromatographed with99:1, 49:1, 24:1, and 23:2 hexanes:ethyl acetate as the eluant to yield4.31 g (81% yield) of1-(3-tert-butyl-phenyl)-3-methylene-cyclohexanecarboxylic acid2-trimethylsilanyl-ethyl ester as a light yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 7.43 (d, J=1.0 Hz, 1H), 7.25 (m, 3H), 4.82 (s,1H), 4.78 (s, 1H), 4.12 (m, 2H), 3.06 (d, J=13.3 Hz, 1H), 2.52 (d,J=13.3 Hz, 2H), 2.26 (dt, J=13.1 Hz and 4.5 Hz, 1H), 2.05 (m, 1H),1.88-1.59 (broad m, 3H), 1.31 (s, 9H), 0.89 (m, 2H), −0.04 (s, 9H).

Step 4

A 1.0 M solution of tetrabutylammonium fluoride in tetrahydrofuran (15.0mL, 15.0 mmol) was added to1-(3-tert-butyl-phenyl)-3-methylene-cyclohexanecarboxylic acid2-trimethylsilanyl-ethyl ester (2.67 mg, 7.16 mmol). After stirring for16 h, the solution was concentrated, diluted with 10% aqueoushydrochloric acid, and extracted with diethyl ether. The combinedorganic extracts were dried over magnesium sulfate, filtered, andconcentrated to yield 2.09 g (100% yield) of1-(3-tert-butyl-phenyl)-3-methylene-cyclohexanecarboxylic acid as ayellow oil.

¹H NMR (300 MHz, CDCl₃) δ 7.50 (m, 1H), 7.29 (m, 3H), 4.84 (s, 1H), 4.79(s, 1H), 3.06 (d, J=13.3 Hz, 1H), 2.58 (d, J=13.3 Hz, 1H), 2.51-1.20(broad m, 6H), 1.34 (s, 9H).

Step 5

Diphenylphosphoryl azide (0.53 mL, 2.46 mmol) was added to a solution of1-(3-tert-butyl-phenyl)-3-methylene-cyclohexanecarboxylic acid (554 mg,2.03 mmol) and triethylamine (0.43 mL, 3.08 mmol) in toluene (4 mL).After stirring at ambient temperature for 18 h, the solution was placedinto a preheated oil bath at 80° C. Bubbling was observed. Afterstirring for 1 h at 80° C., the bubbling had ceased and the solution wascooled to ambient temperature. 10% aqueous hydrochloric acid was addedand stirred vigorously for 3 h. The aqueous layer was made alkaline withaqueous 3 N NaOH and extracted with methylene chloride. The combinedorganic extracts were dried over magnesium sulfate, filtered, andconcentrated. The residue was flash chromatographed with 99:1:0.1,49:1:0.1, 24:1:0.1, and 23:2:0.2 methylenechloride:methanol:concentrated ammonium hydroxide as the eluant to yield12 mg (2% yield) of 1-(3-tert-butyl-phenyl)-3-methylene-cyclohexylamine.

Method [1] Retention time 1.94 min by HPLC and 2.00 min by MS(M−NH₂=227).

Step 6

Using 1-(3-tert-butyl-phenyl)-3-methylene-cyclohexylamine,[3-[1-(3-tert-Butyl-phenyl)-3-methylene-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-carbamicacid tert-butyl ester,3-Amino-1-[1-(3-tert-butyl-phenyl)-3-methylene-cyclohexylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol,andN-[3-[1-(3-tert-Butyl-phenyl)-3-methylene-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamideare prepared according to essentially the same procedure as described inExample 9, steps 1 and 2.

Method [1] Retention time 2.27 min by HPLC and 2.33 min by MS (M+=543).

Method [1] Retention time 1.65 min by HPLC and 1.70 min by MS (M+=443).

Method [1] Retention time 1.92 min by HPLC and 1.98 min by MS (M+=485).

EXAMPLE 20N-[3-[1-(3-TERT-BUTYL-PHENYL)-4-METHYLENE-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDEStep 1

A solution of 1.6 M n-butyllithium in hexanes (46 mL, 73.6 mmol) wasslowly added to a heterogeneous mixture of methyltriphenylphosphoniumbromide (28.07 g, 78.6 mmol) in tetrahydrofuran (150 mL) at −10° C.After stirring for 1 h, 1,4-dioxa-spiro[4.5]decan-8-one (8.01 g, 51.3mmol) was added. After stirring for 3 h, during which time the solutionwarmed to ambient temperature, acetone was added and the heterogeneousmixture was concentrated. The residue was diluted with 1:1 methylenechloride:ethyl ether, filtered and concentrated. The residue was flashchromatographed with 49:1, 24:1, and 23:2 hexanes:etheyl acetate as theeluant to yield 6.22 g (79% yield) of8-methylene-1,4-dioxa-spiro[4.5]decane as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 4.67 (s, 2H), 3.96 (s, 4H), 2.29 (m, 4H), 1.70(m, 4H).

Step 2

A solution of 8-methylene-1,4-dioxa-spiro[4.5]decane (6.22 g, 40.3 mmol)was stirred in tetrahydrofuran (100 mL) and 10% aqueous hydrochloricacid (100 mL) for 18 h. The solution was extracted with ethyl ether andthe combined organic extracts were dried over magnesium sulfate. Thecombined organic extracts were filtered and concentrated to yield 3.89 g(88% yield) of 4-methylene-cyclohexanone as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 4.89 (s, 2H), 2.47 (m, 8H).

Step 3

A solution of 1.7 M tert-butyllithium in pentane (32.0 mL, 54.4 mmol)was added to a solution of 1-bromo-3-tert-butyl-benzene (5.54 g, 26.0mmol) in tetrahydrofuran (60 mL) at −78° C. After stirring for 1 h,cyclohexanone (2.00 g, 18.2 mmol) in tetrahydrofuran (15 mL) was added.After stirring for 18 h, during which time the solution warmed toambient temperature, the solution was diluted with saturated aqueousammonium chloride and extracted with methylene chloride. The combinedorganic extracts were dried over magnesium sulfate, filtered, andconcentrated. The residue was flash chromatographed with 49:1, 24:1,23:2 hexanes:ethyl acetate as the eluant to yield 3.61 g (81% yield) of1-(3-tert-butyl-phenyl)-4-methylene-cyclohexanol as a yellow oil.

¹H NMR (300 MHz, CDCl₃) δ 7.56 (s, 1H), 7.30 (m, 3H), 4.72 (s, 2H), 2.60(m, 2H), 2.27 (m, 2H), 1.93 (m, 4H), 1.33 (s, 9H).

Step 4

Borontrifluoride-etherate (2.0 mL, 15.7 mmol) was added to a solution of1-(3-tert-butyl-phenyl)-4-methylene-cyclohexanol (3.60 g, 14.7 mmol) andazidotrimethylsilane (4.0 mL, 30.1 mmol) in diethyl ether (30 mL) andplaced into a preheated oil bath at 45° C. After heating at reflux for 4h, the solution was diluted with saturated aqueous ammonium chloride andextracted with diethyl ether. The combined organic extracts were driedover magnesium sulfate, filtered, and concentrated. The residue wasflash chromatographed with 99:1, 49:1, and 24:1 hexanes:ethyl acetate asthe eluant to yield 1.46 g (37% yield) of1-(1-azido-4-methylene-cyclohexyl)-3-tert-butyl-benzene as a clear oil.

¹H NMR (300 MHz, CDCl₃) δ 7.47 (s, 1H), 7.36-7.23 (broad m, 3H), 4.72(s, 2H), 2.48 (m, 2H), 2.28 (m, 2H), 2.13 (m, 2H), 1.96 (m, 2H), 1.34(s, 9H).

Step 5

A solution of 1-(1-azido-4-methylene-cyclohexyl)-3-tert-butyl-benzene(820 mg, 3.04 mmol) in diethyl ether (10 mL) was added to aheterogeneous mixture of lithium aluminum hydride (510 mg, 13.4 mmol) indiethyl ether (10 mL) and was placed into a preheated oil bath at 40° C.After heating at reflux for 24 h, the solution was cooled to ambienttemperature, and celite and sodium sulfate decahydrate was added. Afterstirring for 1 h, the heterogeneous mixture was filtered through celiteto yield 1-(3-tert-butyl-phenyl)-4-methylene-cyclohexylamine.

Method [1] Retention time 1.62 min by HPLC and 1.67 min by MS (M+=244).

Step 6

Using 1-(3-tert-butyl-phenyl)-4-methylene-cyclohexylamine,[3-[1-(3-tert-Butyl-phenyl)-4-methylene-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-carbamicacid tert-butyl ester 29,3-Amino-1-[1-(3-tert-butyl-phenyl)-4-methylene-cyclohexylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol30, andN-[3-[1-(3-tert-Butyl-phenyl)-4-methylene-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide31 are prepared according to essentially the same procedure as describedin Example 9, steps 1 and 2.

Method [1] Retention time 2.40 min by HPLC and 2.47 min by MS (M+=543).

Method [1] Retention time 1.36 min by HPLC and 1.42 min by MS (M+=443).

¹H NMR (300 MHz, CDCl₃) δ 9.10 (broad d, 1H), 8.10 (broad d, 1H), 7.61(s, 1H), 7.40 (broad m, 3H), 6.64 (broad s, 3H), 6.50 (m, 1H), 6.00(broad s, 3H), 4.72 (s, 1H), 3.98 (broad s, 1H), 3.77 (broad s, 1H),2.93 (m, 1H), 2.68 (m, 4H), 2.37 (m, 3H), 2.09 (m, 3H), 1.83 (s, 3H),1.32 (s, 9H).

Method [1] Retention time 2.04 min by HPLC and 2.11 min by MS (M+=485).

EXAMPLE 21 Preparation of[4-[3-ACETYLAMINO-4-(3,5-DIFLUORO-PHENYL)-2-HYDROXY-BUTYLAMINO]-4-(3-TERT-BUTYL-PHENYL)-CYCLOHEXYLIDENE]-ACETICACID METHYL ESTER AND OF[4-[3-ACETYLAMINO-4-(3,5-DIFLUORO-PHENYL)-2-HYDROXY-BUTYLAMINO]-4-(3-TERT-BUTYL-PHENYL)-CYCLOHEXYLIDENE]-ACETICACID ETHYL ESTER

To a solution of methyl diethylphosphonoacetate (0.20 mL, 1.102 mmol) inanhydrous THF (1 mL) was added a 60% dispersion of sodium hydride inmineral oil (0.80 g, 20.0 mmol). Vigourous gas evolution was observedwhile stirring at RT under N₂(g) inlet. After 2 h a solution ofN-[3-[1-(3-tert-butyl-phenyl)-4-oxo-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxyl-propyl]-acetamide(0.291 g, 0.598 mmol) in anhydrous THF (1 mL) was added to the reactionflask. The mixture was allowed to stir for 2 days. The reaction wasquenched with H₂O and extracted with CH₂Cl₂. The organic layer wascollected, dried over anhydrous Na₂SO₄, filtered and concentrated. Thecrude product was purified by flash chromatography, eluting with 5%CH₃OH in CH₂Cl₂ yielding 0.085 g of the conjugated products. HPLCpurification afforded the methyl ester: retention time (min)=1.87,method [1]; ¹H NMR (300 MHz, CD₃OD): δ 7.56 (s, 1H), δ 7.31-7.26 (m,1H), δ 7.24-7.26 (m, 2H), δ 6.70 (d, J=7 Hz, 3H), δ 3.80-3.78 (m, 1H), δ3.70 (s, 3H), δ 3.36-3.33 (m, 1H), δ 2.83-2.77 (m, 3H), δ2.51 (t, 1H), δ2.30 (d, J=4 Hz, 1H), δ 2.24 (d, J=10 Hz, 1H), δ 2.16-2.07 (m, 1H), δ1.95-1.86 (m, 7H), δ 1.71 (s, 3H), δ 1.33 (s, 9H); MS (ESI) 543.2 (M+H).

HPLC purification afforded the ethyl ester: retention time (min)=1.98,method [1]; ¹H NMR (300 MHz, CD₃OD): δ 7.55 (s, 1H), δ 7.31-7.27 (m,1H), δ 7.23 (d, J=4 Hz, 2H), δ 6.70 (d, J=8 Hz, 3H), δ 4.20-4.13 (m,2H), δ 3.82-3.76 (m, 1H), δ 3.36-3.35 (m, 1H), δ 2.82 (d, J=4 Hz, 1H), δ2.78 (s, 2H), δ 2.50 (t, 1H), δ 2.33 (d, J=10 Hz, 1H), δ 2.23 (d, J=10Hz, 1H), δ 2.16-2.07 (m, 1H), δ 1.95-1.80 (m, 7H), δ 1.70 (s, 3H), δ1.32 (s, 9H), δ 1.30 (t, 3H); MS (ESI) 557.3 (M+H).

EXAMPLE 22 Preparation of2-[4-[3-ACETYLAMINO-4-3,5-DIFLUORO-PHENYL)-2-HYDROXY-BUTYLAMINO]-4-(3-TERT-BUTYL-PHENYL)-CYCLOHEXYLIDENE]-N,N-DIMETHYL-ACETAMIDE

To a solution of dioctyl (N,N-dimethylcarbamoylmethyl)phosphonate (0.05g, 0.128 mmol) in anhydrous THF (1 mL) was added a 60% dispersion ofsodium hydride in mineral oil (0.026 g, 0.65 mmol). Vigorous gasevolution was observed while stirring at room temperature under N₂(g)inlet. After 3.5 h a solution ofN-[3-[1-(3-tert-butyl-phenyl)-4-oxo-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxyl-propyl]-acetamide(0.041 g, 0.084 mmol) in anhydrous THF (1 mL) was added to the reactionflask. The mixture was allowed to stir overnight. The reaction wasquenched with H₂O and extracted with CH₂Cl₂. The organic layer wascollected, dried over anhydrous sodium sulfate, filtered andconcentrated. HPLC purification afforded the parent compound: retentiontime (min)=1.83, method [1]; ¹H NMR (300 MHz, CD₃OD): δ 7.53 (s, 1H), δ7.24 (m, 1H), 7.22 (d, J=4 Hz, 2H), 6.68 (d, J=8 Hz, 3H), 3.78 (m, 1H),3.41 (m, 1H), 3.12 (s, 3H), 2.94 (s, 3H), 2.84 (s, 2H), 2.79 (d, J=15Hz, 1H), 2.49 (t, 1H), 2.32-2.22 (m, 2H), 2.06 (m, 2H), 1.85 (m, 6H),1.64 (s, 3H), 1.29 (s, 9H); MS (ESI) 556.3 (M+H).

EXAMPLE 23 Preparation ofN-[3-[1-(3-TERT-BUTYL-PHENYL)-4-CYANOMETHYLENE-CYCLOHEXYLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE

To a solution of diethyl(cyanomethyl)phosphonate (0.05 mL, 0.309 mmol)in anhydrous THF (0.7 mL) was added a 60% dispersion of sodium hydridein mineral oil (0.008 g, 0.20 mmol). Vigourous gas evolution wasobserved while stirring at RT under N₂(g) inlet. After 1.5 h a solutionofN-[3-[1-(3-tert-butyl-phenyl)-4-oxo-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxyl-propyl]-acetamide(0.119 g, 0.123 mmol) in anhydrous THF (0.5 mL) was added to thereaction flask. The mixture was allowed to stir overnight. The reactionwas quenched with H₂O and extracted with CH₂Cl₂. The organic layer wascollected, dried over anhydrous Na₂SO₄, filtered and concentrated. Thecrude product was purified by HPLC yielding the trifluoroacetic acidsalt: retention time (min)=1.81, method [1]; MS (ESI) 510.2 (M+H).

EXAMPLE 24 Preparation of Hydrazones

N-[3-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamidewas prepared according to essentially the same procedure as described inEl-Barbary, A. A., J. Heterolytic Chem., 38 (2001), 1711-16.

EXAMPLE 25 Preparation of(2R,3S)-N-[3-[5-(3-TERT-BUTYL-PHENYL)-4,5,6,7-TETRAHYDRO-2H-INDAZOL-5-YLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDEAND(2R,3S)-N-[3-[2-ACETYL-5-(3-TERT-BUTYL-PHENYL)-4,5,6,7-TETRAHYDRO-2H-INDAZOL-5-YLAMINO]-1-(3,5-DIFLUORO-BENZYL)-2-HYDROXY-PROPYL]-ACETAMIDE

Step 1. 4-Amino-4-(3-tert-butyl-phenyl)-cyclohexanone

A solution of 8-(3-tert-Butyl-phenyl)-1,4-dioxa-spiro[4.5]dec-8-ylaminehydrochloride (337 mg, 1.03 mmol) in glacial acetic acid (9 mL) andwater (4 mL) was heated to 75° C. for 21 h, whereupon the reaction wasdeemed complete by HPLC analysis. The reaction mixture was basified with2.5 N NaOH solution, then extracted with ethyl acetate (3×25 mL). Thecombined organic layers were washed (brine), dried (Na₂SO₄), filteredand concentrated under reduced pressure. Material was pure by HPLC/MS:retention time=1.18 min, method [7]; mass spec (ESI) 246 (MH⁺, 33), 229(M−NH₂ ⁺, 100), 228 (28), 173 (47).

Step 2. [1-(3-tert-Butyl-phenyl)-4-oxo-cyclohexyl]-carbamic acidtert-butyl ester

4-Amino-4-(3-tert-butyl-phenyl)-cyclohexanone (441 mg, 1.8 mmol) wasdissolved in dry methylene chloride (10 mL), diisopropylethylamine (0.31mL, 1.8 mmol) and di-tert-butyl carbonate (400 mg, 1.83 mmol) were addedin succession at room temperature. After 19 h, the reaction wasconcentrated under reduced pressure, and the desired product isolated bychromatography (R_(f)=0.35 in 20% EtOAc/hexanes). The resulting oil wastaken to the next reaction: retention time=2.57 min, method [7]; massspec (ESI) 369 (24), 368 (100), 272 (42), 211 (24).

Step 3.[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester

[1-(3-tert-Butyl-phenyl)-4-oxo-cyclohexyl]-carbamic acid tert-butylester (155 mg, 0.45 mmol) was heated with triethylamine (0.010 mL, 0.07mmol) and dimethylformamide dimethyl acetal (0.38 mL, 2.9 mmol) inbenzene (˜20 mL) to reflux in a round-bottom flask fitted with aDean-Stark trap and a condenser. Benzene was distilled to about onethird the original volume, then fresh benzene was added and thedistillation continued. This process was repeated until reaction showedabsence of starting material by TLC (˜24 h). The solvent was evaporated.The crude[1-(3-tert-Butyl-phenyl)-3-dimethylaminomethylene-4-oxo-cyclohexyl]-carbamicacid tert-butyl ester was used in the next reaction without furtherpurification.

To crude[1-(3-tert-Butyl-phenyl)-3-dimethylaminomethylene-4-oxo-cyclohexyl]-carbamicacid tert-butyl ester (91 mg, 0.227 mmol) was added ethanol (6 mL) andhydrazine hydrate (0.010 mL, 0.32 mmol). The reaction mixture wasstirred at room temperature for 12 h then placed in a freezer overnight.The reaction mixture was concentrated under reduced pressure yieldingthe title compound: R_(f)=0.35 in 5% MeOH/CH₂Cl₂; retention time=1.92min, method [7]; mass spec (ESI) 392 (25), 370 (2), 315 (24), 314 (100).

Step 4. 5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamine

[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester (84 mg, 0.23 mmol) was dissolved in 4 N hydrogenchloride in dioxane (2 mL, 8 mmol) at room temperatrure for 90 min,whereupon the reaction was deemed complete by TLC. The reaction mixturewas concentrated under reduced pressure and the amine hydrochloride saltwas basified by partitioning between 1 N aqueous NaOH and 33%isopropanol in chloroform. The organic layer was separated and theaqueous layer extracted twice with additional 33% IPA/CHCl₃. Thecombined organic layers were dried (Na₂SO₄), filtered and concentrated.LC/MS analysis showed 90% pure material, which was taken to subsequentreactions: R_(f)=0.098 in 25% MeOH/CH₂Cl₂; retention time=0.98 min,method [7]; mass spec (ESI) 270 (6), 254 (23), 253 (100), 197 (12).

Step 5.(2R,3S)-[3-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-carbamicacid tert-butyl ester

[2-(3,5-Difluorophenyl)-1-oxiranyl-ethyl]-carbamic acid tert-butyl ester(80.2 mg, 0.268 mmol) was combined with5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamine (61 mg,0.227 mmol) in isopropanol (1 mL). This mixture was heated to 80° C. for16 h, whereupon the reaction mixture was concentrated under reducedpressure and the crude reaction mixture purified by flash chromatographyyielding 61 mg (47%) desired product: R_(f)=0.40 in 10% MeOH/CH₂Cl₂;retention time=1.84 min, method [7]; mass spec (ESI) 591 (20), 570 (42),569 (100).

Step 6.(2R,3S)-N-[3-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamideand(2R,3S)-N-[3-[2-Acetyl-5-(3-tert-butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide

(2R,3S)-[3-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-carbamicacid tert-butyl ester (61 mg, 0.11 mmol) was dissolved in 4 N hydrogenchloride in dioxane (2 mL) at room temperature. After 90 min, thereaction mixture was concentrated under reduced pressure. The cruderesidue was partitioned between 1 N aqueous NaOH and dichloromethane.The layers were separated and the aqueous layer further extracted withdichloromethane twice. The combined organic washes were dried (Na₂SO₄),filtered and concentrated yielding a foam (50 mg), which was used in thesubsequent reaction without further purification.

The crude amine (50 mg, 0.11 mmol) was dissolved in dry dichloromethane(1 mL), and N,N-diacetylhydroxylamine (0.025 mL, 0.21 mmol) was addeddropwise by syringe. After 20 h at room temperature, the reaction wasconcentrated under reduced pressure. The crude residue was purified byHPLC.

(2R,3S)-N-[3-[5-(3-tert-Butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-1-(3,5-difluoro-benzyl)-2-hydroxy-propyl]-acetamide(1:1 mixture of two regioisomers): retention time=1.48 min, method [7];mass spec (ESI) 533 (18), 512 (39), 511 (100), 259 (18), 253 (16).

EXAMPLE 26 NH₂ Replacement of Hydroxyl Alpha to the —(CHR₁)— Group ofCompounds of Formula (I)

EXAMPLE 27 SH Replacement of Hydroxyl Alpha to the —(CHR₁)— Group ofCompounds of Formula (I)

EXAMPLE 28 Preparation of1-[5-(3-TERT-BUTYL-PHENYL)-4,5,6,7-TETRAHYDRO-2H-INDAZOL-5-YLAMINO]-4-(3,5-DIFLUORO-PHENYL)-BUTAN-2-OL

Step 1. Preparation of[5-(3-tert-butyl-phenyl-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester

To [5-(3-tert-butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester (0.58 g, 1.58 mmol) in THF (6 mL) cooled to 0° C.was added a 1.0 M solution of potassium tert-butoxide (3.5 mL). After1.5 h a solution of p-toluenesulfonyl chloride (0.54 g, 2.81 mmol) wasadded and the mixture was stirred overnight while warming to ambienttemperature. The reaction mixture was concentrated under reducedpressure, dissolved in CH₂Cl₂ and washed with H₂O followed by sat. NaCl(aq). The organic layer was collected, dried over anhydrous Na₂SO₄,filtered and concentrated. The crude product was purified by flashchromatography, eluting with 30% ethyl acetate in hexane to afford 0.63g (1.20 mmol, 76%) of[5-(3-tert-butyl-phenyl-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester: retention time (min)=3.18, method [1]. MS (ESI)524.2 (M+H).

Step 2. Preparation of5-(3-tert-butyl-phenyl)-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamine

To a solution of[5-(3-tert-butyl-phenyl-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester (0.61 g, 1.17 mmol) dissolved in CH₂Cl₂ (1 mL) wasadded trifluoacetic acid (1 mL). The reaction mixture was allowed tostir for 1 h prior to quenching with sat. NaHCO₃ (aq) and conc. NH₄OH.The product was extracted with CH₂Cl₂ and the organic layer wascollected, dried over anhydrous Na₂SO₄, filtered and concentrated toafford 0.41 g of crude5-(3-tert-butyl-phenyl)-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamine:retention time of diastereomers (min)=1.62 and 1.71, method [1]. MS(ESI) 407.4 (M+H).

Step 3. Preparation of1-[5-(3-tert-butyl-phenyl)-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol

To a sealed tube was added a solution of5-(3-tert-butyl-phenyl)-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamine(0.41 g, 1.0 mmol) in isopropyl alcohol followed by2-[2-(3,5-difluoro-phenyl)-ethyl]-oxirane (0.18 g, 1.0 mmol). Thereaction was heated at 50° C. overnight and no product was formed. Afterheating at 100° C. for an additional 3.5 h the reaction was complete.The reaction mixture was concentrated and the crude product was purifiedby flash chromatography, eluting with 3% methanol in CHCl₃ to afford0.25 g (0.41 mmol, 42%) of1-[5-(3-tert-butyl-phenyl)-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol:retention time (min)=2.15, method [1]. MS (ESI) 608.4 (M+H).

Step 4. Preparation of1-[5-(3-tert-butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol

To a solution of5-(3-tert-butyl-phenyl)-2-(toluene-4-sulfonyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylaminein methanol (4 mL) was added 1 N NaOH (1 mL). The reaction mixture wasstirred at ambient temperature 5.5 h prior to quenching with H₂O. Theproduct was extracted with EtOAc and the organic layer was collected,dried over anhydrous NaSO₄, filtered and concentrated. The crude productwas purified by flash chromatography, eluting solvent with 5% methanolin CH₂Cl₂ and NH₄OH to afford1-[5-(3-tert-butyl-phenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol.HPLC purification afforded the parent compound: retention time(min)=4.77, method [1]; MS (ESI) 454.2 (M+H).

EXAMPLE 29 Preparation of1-[5-(3-TERT-BUTYL-PHENYL)-2-METHYL-4,5,6,7-TETRAHYDRO-2H-INDAZOL-5-YLAMINO]-4-(3,5-DIFLUORO-PHENYL)-BUTAN-2-OL

Step 1. Preparation of5-(3-tert-butyl-phenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamine

To a solution of[5-(3-tert-butyl-phenyl-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-yl]-carbamicacid tert-butyl ester (0.33 g, 0.86 mmol) dissolved in CH₂Cl₂ (1 mL) wasadded trifluoacetic acid (1 mL). The reaction mixture was allowed tostir for 1.5 h prior to quenching with sat. NaHCO₃ (aq) and conc. NH₄OH.The product was extracted with CH₂Cl₂ and the organic layer wascollected, dried over anhydrous Na₂SO₄, filtered and concentrated toafford 0.17 g of crude5-(3-tert-butyl-phenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamine:retention time (min)=1.04, method [1]. MS (ESI) 267.2 (M+H).

Step 2. Preparation of1-[5-(3-tert-butyl-phenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol

To a sealed tube was added 2-[2-(3,5-difluoro-phenyl)-ethyl]-oxirane(0.124 g, 0.673 mmol) and5-(3-tert-butyl-phenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamine(0.174 g, 0.614 mmol) in a solution of isopropyl alcohol (2.5 mL). Thereaction mixture was heated at 120° C. for 4 h and concentrated to yieldcrude product. The crude product was purified by flash chromatography,eluting with 50% hexane in ethyl acetate, 5% CH₃OH in CH₂Cl₂ and conc.NH₄OH to afford 0.089 g (0.19 mmol, 31%) of the1-[5-(3-tert-butyl-phenyl)-2-methyl-4,5,6,7-tetrahydro-2H-indazol-5-ylamino]-4-(3,5-difluoro-phenyl)-butan-2-ol2. HPLC purification afforded the trifluoroacetic acid salt: retentiontime (min)=1.80, method [1]; ¹H NMR (300 MHz, CD₃OD): δ 7.60 (d, J=2 Hz,1H), δ 7.59-7.27 (m, 4H), δ 6.81-6.75 (m, 3H), δ 3.81 (s, 3H), δ 3.75(m, 1H), δ 3.70 (s, 2H), δ 3.09-3.02 (m, 2H), δ 2.88-2.04 (m, 6H), δ1.71-1.61 (m, 1H), δ 1.37 (m, 2H), δ 1.28 (s, 9H); MS (ESI) 468.3 (M+H).

EXAMPLE 30 Preparation of5-(4-BROMOTHIOPHEN-2-YL)-4,5,6,7-TETRAHYDRO-2H-INDAZOL-5-AMINE

Step 1: Preparation of tert-butyl1-(4-bromothiophen-2-yl)-4-(1,3-dioxol-2-yl)cyclohexylcarbamate

To a stirred solution under nitrogen of 2,4-dibromothiophene (2.24 g,9.25 mmol) in Et₂O (4 mL) at 0° C. was added isopropylmagnesium chloride(2.0 M, 4.78 mL, 9.56 mmol). The reaction was stirred for 30 min to formthe grignard reagent. After this time the reaction was cooled to −78° C.and a solution of 2-Methylpropane-2-sulfinic acid(1,4-dioxa-spiro[4.5]dec-8-ylidene)-amide in Et₂O (and minimal toluenefor solubility) was added to the reaction. Upon addition of the2-Methylpropane-2-sulfinic acid(1,4-dioxa-spiro[4.5]dec-8-ylidene)-amide the reaction was allowed towarm to room temperature and stirred for 3 h. The reaction was thencooled to 0° C. and quenched with water. This mixture was extracted withEtOAc (2×) and the organic phase washed with brine and dried over MgSO₄.The crude product was analyzed by TLC (100% EtOAc), LC and LC/MS. Thesolution was then concentrated and purified on the Biotage Horizon (65+Msilica gel, 10% to 100% EtOAC/Hex51 mL-1836 mL, 100% EtOAc 51 mL-612 mL,100% EtOAc 51 mL-1224 mL). The appropriate fractions were combined (lasteluting) and concentrated to give the thiophene product as a colorlessfoam in 72% yield: ¹H NMR (CDCl₃) δ7.18 (s, 1H), 7.03 (s, 1H), 4.00-3.90(m, 4H), 3.43 (s, 1H), 2.43-2.20 (m, 4H), 1.95-1.65 (m, 4H), 1.17 (s,9H); retention time (min)=3.66 (method [8]); MS (ESI) 122.0.

Step 2: 4-amino-4-(4-bromothiophen-2-yl)-1(1,3-dioxol-2-yl)cyclohexane

To the sulfonylated amine (983 mg, 2.22 mmol) was added HCl (4.0 M inp-dioxane, 5.56 mL, 22.2 mmol) and p-dioxane (4 mL). The reaction wasstirred for 2 h at room temperature and monitored by LC/MS. Followingaddition of the acid a white precipitate formed and persisted for theremainder of the reaction. After this time the reaction was concentratedto a pale yellow oil to afford the deprotected product as the HCl salt.The product was used crude in step 3: retention time (min)=2.47 (method[8]); MS (ESI) 300.9.

Step 3: 4-amino-4-(4-bromothiophen-2-yl)cyclohexanone

To the amine (837 mg, 2.63 mmol) was added an 80:20 solution of AcOH:H₂O(11.5 mL). The reaction was heated to 75° C. and allowed to stir at thistemperature for 2 h. After LC/MS analysis the reaction was allowed tocool to room temperature and then concentrated. The resulting paleorange solid was then re-dissolved in EtOAc and brought to a basic pH(10) with 2M NaOH. The aqueous phase was then extracted with anadditional amount of EtOAc, and all organics combined, dried over MgSO₄,and concentrated to afford the crude product in 81% yield: retentiontime (min)=1.47 (method [8]); MS (ESI) 273.9 (⁷⁹Br isotope).

Step 4: tert-butyl 1-(4-bromothiophen-2-yl)-4-oxocyclohexylcarbamate

To a stirred solution of the ketone (584 mg, 2.13 mmol) in DCM (8 mL)was added Boc anhydride (697 mg, 3.20 mmol). The reaction was allowed tostir at room temperature overnight. The following morning the reactionwas analyzed by TLC (4:1 Hex/EtOAc, MeOH/DCM) and LC/MS. Based on theseresults more Boc anhydride was added (1 eq) and the reaction allowed tocontinue stirring overnight. After this time the reaction wasconcentrated and purified on the Biotage Horizon (40+M silica gel). Theappropriate fractions were combined (TLC 4:1 Hex/EtOAc), concentrated togive the desired product in 82% yield: ¹H NMR (CDCl₃) δ7.07 (s, 1H),6.90 (s, 1H), 4.98 (s, 1H), 2.82-2.65 (m, 2H), 2.65-2.50 (m, 2H),2.45-2.35 (m, 2H), 2.30-2.20 (m, 2H), 1.41 (s, 9H).

Step 5: (E)-tert-butyl1-(4-bromothiophen-2-yl)-3-((dimethylamino)methylene)-4-oxocyclohexylcarbamate

To the ketone (657 mg, 1.76 mmol) in toluene (20 mL), at roomtemperature under nitrogen, was addedtert-butoxybis(dimethylamino)methane (0.4 mL, 1.93 mmol). The reactionwas then heated to 80° C. and left to stir overnight. The followingmorning the reaction was analyzed by TLC and determined to have gone tocompletion. The reaction was allowed to cool, then concentrated and useddirectly in Step 6: retention time (min)=2.08 (method [8]); MS (ESI)430.9.

Step 6: tert-butyl5-(4-bromothiophen-2-yl)-4,5,6,7-tetrahydro-2H-indazol-5-ylcarbamate

To a stirred solution of the enamine (557 mg, 1.91 mmol) in EtOH (20 mL)at room temperature was added hydrazine (119 μl, 3.81 mmol). Thereaction was allowed to stir overnight and monitored by LC/MS and TLC(10% MeOH in DCM). The following morning the reaction was concentratedand then purified on the Biotage Horizon (40+M silica gel, 5% to 75%B:20% MeOH/DCM, 21 mL-1512 mL). The appropriate fractions were combinedand concentrated to give a yellow foam in 73% yield: retention time(min)=2.17 (method [8]); MS (ESI) 399.8.

Step 7: 5-(4-bromothiophen-2-yl)-4,5,6,7-tetrahydro-2H-indazol-5-amine

To a stirred solution of the indazole (557 mg, 1.40 mmol) in p-dioxane(2 mL) was added HCl (4.0M in p-dioxane, 1.75 mL, 6.99 mmol). Thereaction was allowed to stir for 2 h and monitored by TLC (20%MeOH/DCM), LC and LC/MS. After this time the reaction was concentratedand placed on the high vacuum for 48 h. After this time the reaction wasanalyzed by LC and LC/MS and determined not to have gone to completion.Therefore, p-dioxane (2 mL) and HCl (4.0M in p-dioxane, 1.75 mL, 6.99mmol) were added to the dried reaction and allowed to stir for anadditional two h. The reaction was then concentrated, triturated withether/DCM, and the desired product collected as a light orangish yellowsolid in 93% yield: ¹H NMR (DMSO-d₆) δ8.88 (s, 1H), 7.65 (s, 1H), 7.52(s, 1H), 7.38 (s, 1H), 4.80 (br s, 2H), 3.33 (d, J=14 Hz, 1H), 3.14 (d,J=14 Hz, 1H), 2.85-2.72 (m, 1H), 2.50-2.30 (m, 3H); retention time(min)=2.20 (method [8]); MS (ESI) 300.1 (60), 283.1 (⁸¹Br, 100).

EXAMPLE 31 Preparation of5-(3-TERT-BUTYLPHENYL)-4,5,6,7-TETRAHYDRO-2H-INDAZOL-5-AMINE

Step 1: (E)-tert-butyl1-(3-tert-butylphenyl)-3-((dimethylamino)methylene)-4-oxocyclohexylcarbamate

To a stirred solution of tert-butyl1-(3-tert-butylphenyl)-4-oxocyclohexylcarbamate (691 mg, 2.0 mmol) inToluene (5 mL) was added tert-butoxybis(dimethylamino)methane (454 μL,2.2 mmol). The reaction was stirred at 85° C. overnight. The reactionwas concentrated and used crude in the next step.

Step 2: Tert-butyl5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylcarbamate

To a stirred solution of (E)-tert-butyl1-(3-tert-butylphenyl)-3-((dimethylamino)methylene)-4-oxocyclohexylcarbamate (801 mg, 2.0 mmol) in 6 mL ofethanol was added hydrazine monohydrate (150 μL, 3.0 mmol). The reactionwas allowed to stir for 3 h. The reaction was concentrated and purifiedusing a biotage 40S column eluting with DCM/MeOH (95:5) to afford 500 mg(67% yield) of an off white foam. LCMS corresponded to the desiredmaterial: retention time (min)=2.45 (method [8]); MS (ESI) 370.0.

Step 3: 5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-amine

Tert-butyl5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylcarbamate (499mg, 1.35 mmol) was taken up in 2 mL of dioxane followed by the additionof HCl (3.38 mL of a 4N solution in dioxane). The reaction was stirredfor 4 h and then concentrated to yield 350 mg (96% yield) of a whitesolid. ¹H-NMR (CD₃OD) δ8.17 (d, J=2.7 Hz, 1H), 7.60 (s, 1H), 7.52-7.45(m, 1H), 7.44-7.32 (m, 2H), 3.77 (d, J=14.8 Hz, 1H), 3.21 (d, J=14.8 Hz,1H), 3.10-2.94 (m, 1H), 2.82-2.66 (m, 1H), 2.59-2.42 (m, 2H), 1.30 (s,9H); retention time (min)=1.70 (method [8]); MS (ESI) 253.3.

Generally, the protection of amines is conducted, where appropriate, bymethods known to those skilled in the art. See, for example, ProtectingGroups in Organic Synthesis, John Wiley and Sons, New York, N.Y., 1981,Chapter 7; Protecting Groups in Organic Chemistry, Plenum Press, NewYork, N.Y., 1973, Chapter 2. When the amino protecting group is nolonger needed, it is removed by methods known to those skilled in theart. By definition the amino protecting group must be readily removable.A variety of suitable methodologies are known to those skilled in theart; see also T. W. Green and P. G. M. Wuts in Protective Groups inOrganic Chemistry, John Wiley and Sons, 3^(rd) edition, 1999. Suitableamino protecting groups include t-butoxycarbonyl, benzyl-oxycarbonyl,formyl, trityl, phthalimido, trichloro-acetyl, chloroacetyl,bromoacetyl, iodoacetyl, 4-phenylbenzyloxycarbonyl,2-methylbenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl,4-fluorobenzyloxycarbonyl, 4-chlorobenzyloxycarbonyl,3-chlorobenzyloxycarbonyl, 2-chlorobenzyloxycarbonyl,2,4-dichlorobenzyloxycarbonyl, 4-bromobenzyloxycarbonyl,3-bromobenzyloxycarbonyl, 4-nitrobenzyloxycarbonyl,4-cyanobenzyloxycarbonyl, 2-(4-xenyl)isopropoxycarbonyl,1,1-diphenyleth-1-yloxycarbonyl, 1,1-diphenylprop-1-yloxycarbonyl,2-phenylprop-2-yloxycarbonyl, 2-(p-toluyl)prop-2-yloxy-carbonyl,cyclopentanyloxycarbonyl, 1-methylcyclo-pentanyloxycarbonyl,cyclohexanyloxycarbonyl, 1-methyl-cyclohexanyloxycabonyl,2-methylcyclohexanyloxycarbonyl, 2-(4-toluylsulfonyl)ethoxycarbonyl,2-(methylsulfonyl)-ethoxycarbonyl, 2-(triphenylphosphino)ethoxycarbonyl,fluorenylmethoxycarbonyl, 2-(trimethylsilyl)ethoxy-carbonyl,allyloxycarbonyl, 1-(trimethylsilylmethyl)prop-1-enyloxycarbonyl,5-benzisoxalylmethoxycarbonyl, 4-acetoxybenzyloxycarbonyl,2,2,2-trichloroethoxycarbonyl, 2-ethynyl-2-propoxycarbonyl,cyclopropylmethoxycarbonyl, 4-(decyloxyl)benzyloxycarbonyl,isobornyloxycarbonyl, 1-piperidyloxycarbonyl, 9-fluoroenylmethylcarbonate, —CH—CH═CH₂, and the like.

In an embodiment, the protecting group is t-butoxycarbonyl (Boc) and/orbenzyloxycarbonyl (CBZ). In another embodiment, the protecting group isBoc. One skilled in the art will recognize suitable methods ofintroducing a Boc or CBZ protecting group and may additionally consultProtective Groups in Organic Chemistry, for guidance.

The compounds of the present invention may contain geometric or opticalisomers as tautomers. Thus, the present invention includes all tautomersand pure geometric isomers, such as the E and Z geometric isomers, asmixtures thereof. Further, the present invention includes pureenantiomers, diastereomers and/or mixtures thereof, including racemicmixtures. The individual geometric isomers, enantiomers or diastereomersmay be prepared or isolated by methods known to those in the art,including, for example chiral chromatography, preparing diastereomers,separating the diastereomers and then converting the diastereomers intoenantiomers.

Compounds of the present invention with designated stereochemistry canbe included in mixtures, including racemic mixtures, with otherenantiomers, diastereomers, geometric isomers or tautomers. In anotherembodiment, compounds of the present invention are typically present inthese mixtures in diastereomeric and/or enantiomeric excess of at least50%. Compounds of the present invention may be present in these mixturesin diastereomeric and/or enantiomeric excess of at least 80%. Compoundsof the present invention with the desired stereochemistry may also bepresent in diastereomeric and/or enantiomeric excess of at least 90%.Compounds of the present invention with the desired stereochemistry maybe present in diastereomeric and/or enantiomeric excess of at least 99%.The compounds of the present invention may have the “S” configuration atposition 1. Compounds may also have the “R” configuration at position 2.Compounds may, for example, have the “1S,2R” configuration.

All compound names were generated using AutoNom (AUTOmatic NOMenclature)version 2.1, ACD Namepro version 5.09, Chemdraw Ultra (versions 6.0,8.0, 8.03, and 9.0), or were derived therefrom.

Several of the compounds of formula (I) are amines, and as such formsalts when reacted with acids. Pharmaceutically acceptable salts arepreferred over the corresponding amines since they produce compoundswhich are more water soluble, stable and/or more crystalline.

EXAMPLE 32 Biological Examples

Properties such as efficacy, oral bioavailability, selectivity, orblood-brain penetration can be assessed by techniques and assays knownto one skilled in the art. Exemplary assays for determining suchproperties are found below.

Inhibition of APP Cleavage

The methods of treatment and compounds of the present invention inhibitcleavage of APP between Met595 and Asp596 numbered for the APP695isoform, or a mutant thereof, or at a corresponding site of a differentisoform, such as APP751 or APP770, or a mutant thereof (sometimesreferred to as the “beta secretase site”). While many theories exist,inhibition of beta-secretase activity is thought to inhibit productionof A-beta.

Inhibitory activity is demonstrated in one of a variety of inhibitionassays, whereby cleavage of an APP substrate in the presence ofbeta-secretase enzyme is analyzed in the presence of the inhibitorycompound, under conditions normally sufficient to result in cleavage atthe beta-secretase cleavage site. Reduction of APP cleavage at thebeta-secretase cleavage site compared with an untreated or inactivecontrol is correlated with inhibitory activity. Assay systems that canbe used to demonstrate efficacy of the compounds of formula (I) areknown. Representative assay systems are described, for example, in U.S.Pat. Nos. 5,942,400 and 5,744,346, as well as in the Examples below.

The enzymatic activity of beta-secretase and the production of A-betacan be analyzed in vitro or in vivo, using natural, mutated, and/orsynthetic APP substrates, natural, mutated, and/or synthetic enzyme, andthe compound employed in the particular method of treatment. Theanalysis can involve primary or secondary cells expressing native,mutant, and/or synthetic APP and enzyme, animal models expressing nativeAPP and enzyme, or can utilize transgenic animal models expressing thesubstrate and enzyme. Detection of enzymatic activity can be by analysisof at least one of the cleavage products, for example, by immunoassay,fluorometric or chromogenic assay, HPLC, or other means of detection.Inhibitory compounds are determined as those able to decrease the amountof beta-secretase cleavage product produced in comparison to a control,where beta-secretase mediated cleavage in the reaction system isobserved and measured in the absence of inhibitory compounds.

Efficacy reflects a preference for a target tissue. For example,efficacy values yield information regarding a compound's preference fora target tissue by comparing the compound's effect on multiple (e.g.,two) tissues. See, for example, Dovey et al., J. Neurochemistry, 2001,76:173-181. Efficacy reflects the ability of compounds to target aspecific tissue and create the desired result (e.g., clinically).Efficacious compositions and corresponding methods of treatment areneeded to prevent or treat conditions and diseases associated withamyloidosis.

Efficacious compounds of the present invention are those able todecrease the amount of A-beta produced compared to a control, wherebeta-secretase mediated cleavage is observed and measured in the absenceof the compounds. Detection of efficacy can be by analysis of A-betalevels, for example, by immunoassay, fluorometric or chromogenic assay,HPLC, or other means of detection. The efficacy of the compounds offormula (I) was determined as a percentage inhibition corresponding toA-beta concentrations for tissue treated and untreated with a compoundof formula (I).

Beta-Secretase

Various forms of beta-secretase enzyme are known, are available, anduseful for assaying of enzymatic activity and inhibition of enzymeactivity. These include native, recombinant, and synthetic forms of theenzyme. Human beta-secretase is known as Beta Site APP Cleaving Enzyme(BACE), BACE1, Asp2, and memapsin 2, and has been characterized, forexample, in U.S. Pat. No. 5,744,346 and published PCT patentapplications WO 98/22597, WO 00/03819, WO 01/23533, and WO 00/17369, aswell as in literature publications (Hussain et al., 1999, Mol. Cell.Neurosci., 14:419-427; Vassar et al., 1999, Science, 286:735-741; Yan etal., 1999, Nature, 402:533-537; Sinha et al., 1999, Nature, 40:537-540;and Lin et al., 2000, Proceedings Natl. Acad. Sciences USA,97:1456-1460). Synthetic forms of the enzyme have also been describedin, for example (WO 98/22597 and WO 00/17369). Beta-secretase can beextracted and purified from human brain tissue and can be produced incells, for example mammalian cells expressing recombinant enzyme.

APP Substrate

Assays that demonstrate inhibition of beta-secretase-mediated cleavageof APP can utilize any of the known forms of APP, including the 695amino acid “normal” isotype described by Kang et al., 1987, Nature,325:733-6, the 770 amino acid isotype described by Kitaguchi et. al.,1981, Nature, 331:530-532, and variants such as the Swedish Mutation(KM670-1NL) (APP-SW), the London Mutation (V7176F), and others. See, forexample, U.S. Pat. No. 5,766,846 and also Hardy, 1992, Nature Genet.1:233-234, for a review of known variant mutations. Additional usefulsubstrates include the dibasic amino acid modification, APP-KK,disclosed, for example, in WO 00/17369, fragments of APP, and syntheticpeptides containing the beta-secretase cleavage site, wild type (WT) ormutated form, (e.g., SW), as described, for example, in U.S. Pat. No.5,942,400 and WO 00/03819.

The APP substrate contains the beta-secretase cleavage site of APP(KM-DA, SEQ ID NO: 1 or NL-DA, SEQ ID NO: 2) for example, a complete APPpeptide or variant, an APP fragment, a recombinant or synthetic APP, ora fusion peptide. Preferably, the fusion peptide includes thebeta-secretase cleavage site fused to a peptide having a moiety usefulfor enzymatic assay, for example, having isolation and/or detectionproperties. A useful moiety can be an antigenic epitope for antibodybinding, a label or other detection moiety, a binding substrate, and thelike.

Antibodies

Products characteristic of APP cleavage can be measured by immunoassayusing various antibodies, as described, for example, in Pirttila et al.,1999, Neuro. Lett., 249:21-4, and in U.S. Pat. No. 5,612,486. Usefulantibodies to detect A-beta include, for example, the monoclonalantibody 6E10 (Senetek, St. Louis, Mo.) that specifically recognizes anepitope on amino acids 1-16 of the A-beta peptide, antibodies 162 and164 (New York State Institute for Basic Research, Staten Island N.Y.)that are specific for human A-beta 1-40 and 1-42, respectively, andantibodies that recognize the junction region of A-beta, the sitebetween residues 16 and 17, as described in U.S. Pat. No. 5,593,846.Antibodies raised against a synthetic peptide of residues 591 to 596 ofAPP and SW192 antibody raised against 590-596 of the Swedish mutationare also useful in immunoassay of APP and its cleavage products, asdescribed in U.S. Pat. Nos. 5,604,102 and 5,721,130.

Assay Systems

Assays for determining APP cleavage at the beta-secretase cleavage siteare well known in the art. Exemplary assays, are described, for example,in U.S. Pat. Nos. 5,744,346 and 5,942,400, and described in the Examplesbelow.

Cell Free Assays

Exemplary assays that can be used to demonstrate the inhibitory activityof the compounds of the present invention are described, for example, inWO 00/17369, WO 00/03819, and U.S. Pat. Nos. 5,942,400 and 5,744,346.Such assays can be performed in cell-free incubations or in cellularincubations using cells expressing a beta-secretase and an APP substratehaving a beta-secretase cleavage site.

An APP substrate containing the beta-secretase cleavage site of APP, forexample, a complete APP or variant, an APP fragment, or a recombinant orsynthetic APP substrate containing the amino acid sequence KM-DA (SEQ IDNO: 1) or NL-DA (SEQ ID NO: 2) is incubated in the presence ofbeta-secretase enzyme, a fragment thereof, or a synthetic or recombinantpolypeptide variant having beta-secretase activity and effective tocleave the beta-secretase cleavage site of APP, under incubationconditions suitable for the cleavage activity of the enzyme. Suitablesubstrates optionally include derivatives that can be fusion proteins orpeptides that contain the substrate peptide and a modification useful tofacilitate the purification or detection of the peptide or itsbeta-secretase cleavage products. Useful modifications include theinsertion of a known antigenic epitope for antibody binding, the linkingof a label or detectable moiety, the linking of a binding substrate, andthe like.

Suitable incubation conditions for a cell-free in vitro assay include,for example, approximately 200 nM to 10 μM substrate, approximately 10pM to 200 pM enzyme, and approximately 0.1 nM to 10 μM inhibitorcompound, in aqueous solution, at an approximate pH of 4-7, atapproximately 37° C., for a time period of approximately 10 min to 3 h.These incubation conditions are exemplary only, and can vary as requiredfor the particular assay components and/or desired measurement system.Optimization of the incubation conditions for the particular assaycomponents should account for the specific beta-secretase enzyme usedand its pH optimum, any additional enzymes and/or markers that might beused in the assay, and the like. Such optimization is routine and willnot require undue experimentation.

One useful assay utilizes a fusion peptide having maltose bindingprotein (MBP) fused to the C-terminal 125 amino acids of APP-SW. The MBPportion is captured on an assay substrate by an anti-MBP captureantibody. Incubation of the captured fusion protein in the presence ofbeta-secretase results in cleavage of the substrate at thebeta-secretase cleavage site. Analysis of the cleavage activity can be,for example, by immunoassay of cleavage products. One such immunoassaydetects a unique epitope exposed at the carboxy terminus of the cleavedfusion protein, for example, using the antibody SW192. This assay isdescribed, for example, in U.S. Pat. No. 5,942,400.

Cellular Assay

Numerous cell-based assays can be used to analyze beta-secretaseactivity and/or processing of APP to release A-beta. Contact of an APPsubstrate with a beta-secretase enzyme within the cell and in thepresence or absence of a compound inhibitor of the present invention canbe used to demonstrate beta-secretase inhibitory activity of thecompound. It is preferred that the assay in the presence of a usefulinhibitory compound provides at least about 10% inhibition of theenzymatic activity, as compared with a non-inhibited control.

In an embodiment, cells that naturally express beta-secretase are used.Alternatively, cells are modified to express a recombinantbeta-secretase or synthetic variant enzyme as discussed above. The APPsubstrate can be added to the culture medium and is preferably expressedin the cells. Cells that naturally express APP, variant or mutant formsof APP, or cells transformed to express an isoform of APP, mutant orvariant APP, recombinant or synthetic APP, APP fragment, or syntheticAPP peptide or fusion protein containing the beta-secretase APP cleavagesite can be used, provided that the expressed APP is permitted tocontact the enzyme and enzymatic cleavage activity can be analyzed.

Human cell lines that normally process A-beta from APP provide usefulmeans to assay inhibitory activities of the compounds employed in themethods of treatment of the present invention. Production and release ofA-beta and/or other cleavage products into the culture medium can bemeasured, for example by immunoassay, such as Western blot orenzyme-linked immunoassay (EIA) such as by ELISA.

Cells expressing an APP substrate and an active beta-secretase can beincubated in the presence of a compound inhibitor to demonstrateinhibition of enzymatic activity as compared with a control. Activity ofbeta-secretase can be measured by analysis of at least one cleavageproduct of the APP substrate. For example, inhibition of beta-secretaseactivity against the substrate APP would be expected to decrease therelease of specific beta-secretase induced APP cleavage products such asA-beta.

Although both neural and non-neural cells process and release A-beta,levels of endogenous beta-secretase activity are low and often difficultto detect by EIA. The use of cell types known to have enhancedbeta-secretase activity, enhanced processing of APP to A-beta, and/orenhanced production of A-beta are therefore preferred. For example,transfection of cells with the Swedish Mutant form of APP (APP-SW), withAPP-KK, or with APP-SW-KK provides cells having enhanced beta-secretaseactivity and producing amounts of A-beta that can be readily measured.

In such assays, for example, the cells expressing APP and beta-secretaseare incubated in a culture medium under conditions suitable forbeta-secretase enzymatic activity at its cleavage site on the APPsubstrate. On exposure of the cells to the compound inhibitor employedin the methods of treatment, the amount of A-beta released into themedium and/or the amount of CTF99 fragments of APP in the cell lysatesis reduced as compared with the control. The cleavage products of APPcan be analyzed, for example, by immune reactions with specificantibodies, as discussed above.

Preferred cells for analysis of beta-secretase activity include primaryhuman neuronal cells, primary transgenic animal neuronal cells where thetransgene is APP, and other cells such as those of a stable 293 cellline expressing APP, for example, APP-SW.

In Vivo Assays: Animal Models

Various animal models can be used to analyze beta-secretase activityand/or processing of APP to release A-beta, as described above. Forexample, transgenic animals expressing APP substrate and beta-secretaseenzyme can be used to demonstrate inhibitory activity of the compoundsof the present invention. Certain transgenic animal models have beendescribed, for example, in U.S. Pat. Nos. 5,877,399, 5,612,486,5,387,742, 5,720,936, 5,850,003, 5,877,015, and 5,811,633, and in Gameset al., 1995, Nature, 373:523. Animals that exhibit characteristicsassociated with the pathophysiology of Alzheimer's disease arepreferred. Administration of the compounds of the present invention tothe transgenic mice described herein provides an alternative method fordemonstrating the inhibitory activity of the compounds. Administrationof the compounds of the present invention in a pharmaceuticallyeffective carrier and via an administrative route that reaches thetarget tissue in an appropriate therapeutic amount is also preferred.

Inhibition of beta-secretase mediated cleavage of APP at thebeta-secretase cleavage site and of A-beta release can be analyzed inthese animals by measuring cleavage fragments in the animal's bodyfluids such as cerebral fluid or tissues. Analysis of brain tissues forA-beta deposits or plaques is preferred.

A: Enzyme Inhibition Assay

The methods of treatment and compounds of the present invention areanalyzed for inhibitory activity by use of the MBP-C125 assay. Thisassay determines the relative inhibition of beta-secretase cleavage of amodel APP substrate, MBP-C125SW, by the compounds assayed as comparedwith an untreated control. A detailed description of the assayparameters can be found, for example, in U.S. Pat. No. 5,942,400.Briefly, the substrate is a fusion peptide formed of maltose bindingprotein (MBP) and the carboxy terminal 125 amino acids of APP-SW, theSwedish mutation. The beta-secretase enzyme is derived from human braintissue as described in Sinha et al., 1999, Nature, 40:537-540 orrecombinantly produced as the full-length enzyme (amino acids 1-501),and can be prepared, for example, from 293 cells expressing therecombinant cDNA, as described in WO 00/47618.

Inhibition of the enzyme is analyzed, for example, by immunoassay of theenzyme's cleavage products. One exemplary ELISA uses an anti-MBP captureantibody that is deposited on precoated and blocked 96-well high bindingplates, followed by incubation with diluted enzyme reaction supernatant,incubation with a specific reporter antibody, for example, biotinylatedanti-SW192 reporter antibody, and further incubation withstreptavidin/alkaline phosphatase. In the assay, cleavage of the intactMBP-C125SW fusion protein results in the generation of a truncatedamino-terminal fragment, exposing a new SW-192 antibody-positive epitopeat the carboxy terminus. Detection is effected by a fluorescentsubstrate signal on cleavage by the phosphatase. ELISA only detectscleavage following Leu596 at the substrate's APP-SW 751 mutation site.

Specific Assay Procedure

Compounds of formula (I) are diluted in a 1:1 dilution series to asix-point concentration curve (two wells per concentration) in one rowof a 96-well plate per compound tested. Each of the test compounds isprepared in DMSO to make up a 10 mM stock solution. The stock solutionis serially diluted in DMSO to obtain a final compound concentration of200 μM at the high point of a 6-point dilution curve. Ten (10) μL ofeach dilution is added to each of two wells on row C of a correspondingV-bottom plate to which 190 μL of 52 mM NaOAc, 7.9% DMSO, pH 4.5 arepre-added. The NaOAc diluted compound plate is spun down to pelletprecipitant and 20 μL/well is transferred to a corresponding flat-bottomplate to which 30 μL of ice-cold enzyme-substrate mixture (2.5 μLMBP-C125SW substrate, 0.03 μL enzyme and 24.5 μL ice cold 0.09% TX100per 30 μL) is added. The final reaction mixture of 200 μM compound atthe highest curve point is in 5% DMSO, 20 μM NaOAc, 0.06% TX100, at pH4.5.

Warming the plates to 37° C. starts the enzyme reaction. After 90 min at37° C., 200 μL/well cold specimen diluent is added to stop the reactionand 20 μL/well was transferred to a corresponding anti-MBP antibodycoated ELISA plate for capture, containing 80 μL/well specimen diluent.This reaction is incubated overnight at 4° C. and the ELISA is developedthe next day after a 2 hour incubation with anti-192SW antibody,followed by Streptavidin-AP conjugate and fluorescent substrate. Thesignal is read on a fluorescent plate reader.

Relative compound inhibition potency is determined by calculating theconcentration of compound that showed a 50% reduction in detected signal(IC₅₀) compared to the enzyme reaction signal in the control wells withno added compound. In this assay, preferred compounds of the presentinvention exhibit an IC₅₀ of less than 50 μM.

B: FP BACE Assay: Cell Free Inhibition Assay Utilizing a Synthetic APPSubstrate

A synthetic APP substrate that can be cleaved by beta-secretase andhaving N-terminal biotin and made fluorescent by the covalent attachmentof Oregon green at the Cys residue is used to assay beta-secretaseactivity in the presence or absence of the inhibitory compounds employedin the present invention. Useful substrates include

Biotin-SEVNL-DAEFRC[oregon green]KK (SEQ ID NO: 3),

Biotin-SEVKM-DAEFRC[oregon green]KK (SEQ ID NO: 4),

Biotin-GLNIKTEEISEISY-EVEFRC[oregon green]KK (SEQ ID NO: 5),

Biotin-ADRGLTTRPGSGLTNIKTEEISEVNL-DAEFRC[oregon green]KK (SEQ ID NO: 6),and

Biotin-FVNQHLCoxGSHLVEALY-LVCoxGERGFFYTPKAC[oregon green]KK (SEQ ID NO:7).

The enzyme (0.1 nM) and test compounds (0.001-100 μM) are incubated inpre-blocked, low affinity, black plates (384 well) at 37° C. for 30 min.The reaction is initiated by addition of 150 mM substrate to a finalvolume of 30 μL/well. The final assay conditions are 0.001-100 μMcompound inhibitor, 0.1 molar sodium acetate (pH 4.5), 150 nM substrate,0.1 nM soluble beta-secretase, 0.001% Tween 20, and 2% DMSO. The assaymixture is incubated for 3 h at 37° C., and the reaction is terminatedby the addition of a saturating concentration of immunopurestreptavidin. After incubation with streptavidin at room temperature for15 min, fluorescence polarization is measured, for example, using a LJLAcqurest (Ex485 nm/Em530 nm).

The activity of the beta-secretase enzyme is detected by changes in thefluorescence polarization that occur when the substrate is cleaved bythe enzyme. Incubation in the presence or absence of compound inhibitordemonstrates specific inhibition of beta-secretase enzymatic cleavage ofits synthetic APP substrate. In this assay, preferred compounds of thepresent invention exhibit an IC₅₀ of less than 50 μM. More preferredcompounds of the present invention exhibit an IC₅₀ of less than 10 μM.Even more preferred compounds of the present invention exhibit an IC₅₀of less than 5 μM.

C: Beta-Secretase Inhibition: P26-P4′SW Assay

Synthetic substrates containing the beta-secretase cleavage site of APPare used to assay beta-secretase activity, using the methods described,for example, in published PCT application WO 00/47618. The P26-P4′SWsubstrate is a peptide of the sequence(biotin)CGGADRGLTTRPGSGLTNIKTEEISEVNLDAEF (SEQ ID NO: 8). The P26-P1standard has the sequence (biotin)CGGADRGLTTRPGSGLTNIKTEEISEVNL (SEQ IDNO: 9).

Briefly, the biotin-coupled synthetic substrates are incubated at aconcentration of from about 0 to about 200 μM in this assay. Whentesting inhibitory compounds, a substrate concentration of about 1.0 μMis preferred. Test compounds diluted in DMSO are added to the reactionmixture, with a final DMSO concentration of 5%. Controls also contain afinal DMSO concentration of 5%. The concentration of beta secretaseenzyme in the reaction is varied, yielding product concentrations withthe linear range of the ELISA assay, about 125 to 2000 pM, afterdilution.

The reaction mixture also includes 20 mM sodium acetate, pH 4.5, 0.06%Triton X100, and is incubated at 37° C. for about 1 to 3 h. Samples arethen diluted in assay buffer (for example, 145.4 nM sodium chloride,9.51 mM sodium phosphate, 7.7 mM sodium azide, 0.05% Triton X405, 6 g/Lbovine serum albumin, pH 7.4) to quench the reaction, then dilutedfurther for immunoassay of the cleavage products.

Cleavage products can be assayed by ELISA. Diluted samples and standardsare incubated in assay plates coated with capture antibody, for example,SW192, for about 24 h at 4° C. After washing in TTBS buffer (150 mMsodium chloride, 25 mM Tris, 0.05% Tween 20, pH 7.5), the samples areincubated with streptavidin-AP according to the manufacturer'sinstructions. After a 1 h incubation at room temperature, the samplesare washed in TTBS and incubated with fluorescent substrate solution A(31.2 g/L 2-amino-2-methyl-1-propanol, 30 mg/L, pH 9.5). Reaction withstreptavidin-alkaline phosphate permits detection by fluorescence.Compounds that are effective inhibitors of beta-secretase activitydemonstrate reduced cleavage of the substrate as compared to a control.

D: Assays Using Synthetic Oligopeptide-Substrates

Synthetic oligopeptides are prepared incorporating the known cleavagesite of beta-secretase, and optionally include detectable tags, such asfluorescent or chromogenic moieties. Examples of such peptides, as wellas their production and detection methods, are described in U.S. Pat.No. 5,942,400. Cleavage products can be detected using high performanceliquid chromatography, or fluorescent or chromogenic detection methodsappropriate to the peptide to be detected, according to methods wellknown in the art.

By way of example, one such peptide has the sequence SEVNL-DAEF (SEQ IDNO: 10), and the cleavage site is between residues 5 and 6. Anotherpreferred substrate has the sequence ADRGLTTRPGSGLTNIKTEEISEVNL-DAEF(SEQ ID NO: 11), and the cleavage site is between residues 26 and 27.

These synthetic APP substrates are incubated in the presence ofbeta-secretase under conditions sufficient to result in beta-secretasemediated cleavage of the substrate. Comparison of the cleavage resultsin the presence of a compound inhibitor to control results provides ameasure of the compound's inhibitory activity.

E: Inhibition of Beta-Secretase Activity-Cellular Assay

An exemplary assay for the analysis of inhibition of beta-secretaseactivity utilizes the human embryonic kidney cell line HEKp293 (ATCCAccession No. CRL-1573) transfected with APP751 containing the naturallyoccurring double mutation Lys651Met652 to Asn651Leu652 (numbered forAPP751), commonly called the Swedish mutation and shown to overproduceA-beta (Citron et al., 1992, Nature, 360:672-674), as described in U.S.Pat. No. 5,604,102.

The cells are incubated in the presence/absence of the inhibitorycompound (diluted in DMSO) at the desired concentration, generally up to10 μg/mL. At the end of the treatment period, conditioned media isanalyzed for beta-secretase activity, for example, by analysis ofcleavage fragments. A-beta can be analyzed by immunoassay, usingspecific detection antibodies. The enzymatic activity is measured in thepresence and absence of the compounds of formula (I) to demonstratespecific inhibition of beta-secretase mediated cleavage of APPsubstrate.

F: Inhibition of Beta-Secretase in Animal Models of Alzheimer's Disease

Various animal models can be used to screen for inhibition ofbeta-secretase activity. Examples of animal models useful in the presentinvention include mouse, guinea pig, dog, and the like. The animals usedcan be wild type, transgenic, or knockout models. In addition, mammalianmodels can express mutations in APP, such as APP695-SW and the like asdescribed herein. Examples of transgenic non-human mammalian models aredescribed in U.S. Pat. Nos. 5,604,102, 5,912,410 and 5,811,633.

PDAPP mice, prepared as described in Games et al., 1995, Nature,373:523-527 are useful to analyze in vivo suppression of A-beta releasein the presence of putative inhibitory compounds. As described in U.S.Pat. No. 6,191,166, 4-month-old PDAPP mice are administered a compoundof formula (I) formulated in a vehicle, such as corn oil. The mice aredosed with the compound (1-30 mg/mL, preferably 1-10 mg/mL). After adesignated time, e.g., 3-10 h, the brains are analyzed.

Transgenic animals are administered an amount of a compound formulatedin a carrier suitable for the chosen mode of administration. Controlanimals are untreated, treated with vehicle, or treated with an inactivecompound. Administration can be acute, (i.e. single dose or multipledoses in one day), or can be chronic, (i.e. dosing is repeated daily fora period of days). Beginning at time 0, brain tissue or cerebral fluidis obtained from selected animals and analyzed for the presence of APPcleavage peptides, including A-beta, for example, by immunoassay usingspecific antibodies for A-beta detection. At the end of the test period,animals are sacrificed and brain tissue or cerebral fluid is analyzedfor the presence of A-beta and/or beta-amyloid plaques. The tissue isalso analyzed for necrosis.

Reduction of A-beta in brain tissues or cerebral fluids and reduction ofbeta-amyloid plaques in brain tissue are assessed by administering thecompounds of formula (I), or pharmaceutical compositions comprisingcompounds of formula (I) to animals and comparing the data with thatfrom non-treated controls.

G: Inhibition of A-Beta Production in Human Patients

Patients suffering from Alzheimer's disease demonstrate an increasedamount of A-beta in the brain. Alzheimer's disease patients aresubjected to a method of treatment of the present invention, (i.e.administration of an amount of the compound inhibitor formulated in acarrier suitable for the chosen mode of administration). Administrationis repeated daily for the duration of the test period. Beginning on day0, cognitive and memory tests are performed, for example, once permonth.

Patients administered the compounds of formula (I) are expected todemonstrate slowing or stabilization of disease progression as analyzedby a change in at least one of the following disease parameters: A-betapresent in cerebrospinal fluid or plasma; brain or hippocampal volume;A-beta deposits in the brain; amyloid plaque in the brain; or scores forcognitive and memory function, as compared with control, non-treatedpatients.

H: Prevention of A-Beta Production in Patients at Risk for Alzheimer'sDisease

Patients predisposed or at risk for developing Alzheimer's disease canbe identified either by recognition of a familial inheritance pattern,for example, presence of the Swedish Mutation, and/or by monitoringdiagnostic parameters. Patients identified as predisposed or at risk fordeveloping Alzheimer's disease are administered an amount of thecompound inhibitor formulated in a carrier suitable for the chosen modeof administration. Administration is repeated daily for the duration ofthe test period. Beginning on day 0, cognitive and memory tests areperformed, for example, once per month.

Patients subjected to a method of treatment of the present invention(i.e., administration of at least one compound of formula (I)) areexpected to demonstrate slowing or stabilization of disease progressionas analyzed by a change in at least one of the following diseaseparameters: A-beta present in cerebrospinal fluid or plasma; brain orhippocampal volume; amyloid plaque in the brain; or scores for cognitiveand memory function, as compared with control, non-treated patients.

I: Efficacy of Compounds to Inhibit A-Beta Concentration

The invention encompasses compounds of formula (I) that are efficacious.Efficacy is calculated as a percentage of concentrations as follows:Efficacy=(1−(total A-beta in dose group/total A-beta in vehiclecontrol))*100%wherein the “total A-beta in dose group” equals the concentration ofA-beta in the tissue, (e.g., rat brain) treated with the compound, andthe “total A-beta in vehicle control” equals the concentration of A-betain the tissue, yielding a % inhibition of A-beta production. Statisticalsignificance is determined by p-value<0.05 using the Mann Whitneyt-test. See, for example, Dovey et al., J. Neurochemistry, 2001,76:173-181.

Where indicated, diastereomers were separated by reverse phase HPLCusing the noted methods. The first isomer collected in each case wasdesignated Diastereomer A, and the second isomer Diastereomer B. Unlessotherwise indicated, specific formula (I) compound examples representmixtures of diastereomers.

J: Selectivity of Compounds for Inhibiting BACE Over Aspartyl Proteases

The compounds of formula (I) can be selective for beta-secretase versuscatD. Wherein the ratio of catD:beta-secretase is greater than 1,selectivity is calculated as follows:Selectivity=(IC₅₀ for catD/IC₅₀ for beta-secretase)*100%wherein IC₅₀ is the concentration of compound necessary to decrease thelevel of catD or beta-secretase by 50%.

The compounds of formula (I) can be selective for beta-secretase versuscatE. Wherein the ratio of catE:beta-secretase is greater than 1,selectivity is calculated as follows:Selectivity=(IC₅₀ for catE/IC₅₀ for beta-secretase)*100%wherein IC₅₀ is the concentration of compound necessary to decrease thelevel of catE or beta-secretase by 50%. Selectivity is reported as theratio of IC₅₀(catE):IC₅₀(BACE).

Pharmacokinetic parameters were calculated by a non-compartmentalapproach. See, for example, Gibaldi, M. and Perrier, D.,Pharmacokinetics, Second Edition, 1982, Marcel Dekker Inc., New York,N.Y., pp 409-418.

EXAMPLE 33 Exemplary Formula (I) Compounds Exhibiting Selectivity forBace Versus catD

In the following examples, each value is an average of four experimentalruns and multiple values for one compound indicate that more than oneexperiment was conducted.

Ex- am- Selectivity ple IC₅₀(catD)/ No. Compound IC₅₀(BACE) 33-1

1.8>2.4  1.01-(5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino)-4-(3,5-difluorophenyl)butan-2-ol

EXAMPLE 34 Exemplary Formula (I) Compounds Exhibiting Selectivity forBace Versus catE

Ex- am- Selectivity ple IC₅₀(catE)/ No. Compound IC₅₀(BACE) 34-1

1.61.60.61-(5-(3-tert-butylphenyl)-4,5,6,7-tetrahydro-2H-indazol-5-ylamino)-4-(3,5-difluorophenyl)butan-2-ol

Oral Bioavailability of Compounds for Inhibiting Amyloidosis

The invention encompasses compounds of formula (I) that are orallybioavailable. Generally, oral bioavailability is defined as the fractionof orally administered dose reaching systemic circulation. Oralbioavailability can be determined following both an intravenous (IV) andoral (PO) administration of a test compound.

Oral bioavailability was determined in the male Sprague-Dawley ratfollowing both IV and PO administration of test compound. Two month-oldmale rats (250-300 g) were surgically implanted with polyethylene(PE-50) cannula in the jugular vein while under isoflurane anesthesiathe day before the in-life phase. Animals were fasted overnight withwater ad libitum, then dosed the next day. The dosing regime consistedof either a 5 mg/kg (2.5 mL/kg) IV dose (N=3) administered to thejugular vein cannula, then flushed with saline, or a 10 mg/kg (5 mL/kg)PO dose (N=3) by esophageal gavage. Compounds were formulated with 10%Solutol in 5% dextrose at 2 mg/mL. Subsequent to dosing, blood wascollected at 0.016 (IV only), 0.083, 0.25, 0.5, 1, 3, 6, 9 and 24 h postadministration and heparinized plasma was recovered followingcentrifugation.

Compounds were extracted from samples following precipitation of theplasma proteins by methanol. The resulting supernatants were evaporatedto dryness and reconstituted with chromatographic mobile phase (35%acetonitrile in 0.1% formic acid) and injected onto a reverse phase C₁₈column (2×50 mm, 5 μm, BDS Hypersil). Detection was facilitated with amulti-reaction-monitoring experiment on a tandem triple quadrupole massspectrometer (LC/MS/MS) following electrospray ionization. Experimentalsamples were compared to calibration curves prepared in parallel withaged match rat plasma and quantitated with a weighted 1/x linearregression. The lower limit of quantization (LOQ) for the assay wastypically 0.5 ng/mL.

Oral bioavailability (% F or F value) is calculated from thedose-normalized ratio of plasma exposure following oral administrationto the intravenous plasma exposure in the rat by the following equation% F=(AUC_(po)/AUC_(iv))×(D _(iv) /D _(po))×100%where D is the dose and AUC is thearea-under-the-plasma-concentration-time-curve from 0 to 24 h. AUC iscalculated from the linear trapezoidal rule by AUC=((C₂+C₁)/2)×(T₂−T₁)where C is concentration and T is time.

Pharmacokinetic parameters were calculated by a non-compartmentalapproach. See, for example, Gibaldi, M. and Perrier, D.,Pharmacokinetics, Second Edition, 1982, Marcel Dekker Inc., New York,N.Y., pp 409-418.L: Brain Uptake

The invention encompasses beta-secretase inhibitors that can readilycross the blood-brain barrier. Factors that affect a compound's abilityto cross the blood-brain barrier include a compound's molecular weight,Total Polar Surface Area (TPSA), and log P (lipophilicity). See, e.g.,Lipinski, C. A., et al., Adv. Drug Deliv. Reviews, 23:3-25 (1997). Oneof ordinary skill in the art will be aware of methods for determiningcharacteristics allowing a compound to cross the blood-brain barrier.See, for example, Murcko et al., Designing Libraries with CNS Activity,J. Med. Chem., 42 (24), pp. 4942-51 (1999). Calculations of logP valueswere performed using the Daylight clogP program (Daylight ChemicalInformation Systems, Inc.). See, for example, Hansch, C., et al.,Substituent Constants for Correlation Analysis in Chemistry and Biology,Wiley, New York (1979); Rekker, R., The Hydrophobic Fragmental Constant,Elsevier, Amsterdam (1977); Fujita, T., et al., J. Am. Chem. Soc., 86,5157 (1964). TPSA was calculated according to the methodology outlinedin Ertl, P., et al., J. Med. Chem., 43:3714-17 (2000).

The following assay was employed to determine the brain penetration ofcompounds encompassed by the present invention.

In-life phase: Test compounds were administered to CF-1 (20-30 g) miceat 10 μmol/kg (4 to 7 mg/kg) following IV administration in the tailvein. Two time-points, 5 and 60 min, are collected post dose. Four miceare harvested for heparinized plasma and non-perfused brains at eachtime-point for a total of 8 mice per compound.

Analytical phase: Samples were extracted and evaporated to dryness, thenreconstituted and injected onto a reverse phase chromatographic columnwhile monitoring the effluent with a triple quadrupole massspectrometer. Quantitation was then performed with a 1/x² weighted fitof the least-squares regression from calibration standards prepared inparallel with the in vivo samples. The LOQ is generally 1 ng/mL and 0.5ng/g for the plasma and brain respectively. Data was reported inmicromolar (μM) units. Brain levels were corrected for plasma volumes(16 μL/g).

Results: Comparison of a compound's brain concentration level to twomarker compounds, Indinavir and Diazepam, demonostrates the ability inwhich the compounds of the present invention can cross the blood-brainbarrier. Indinavir (HIV protease inhibitor) is a poor brain penetrantmarker and Diazepam is a blood flow limited marker. The concentrationlevels of Indinavir in the brain at 5 and 60 min were 0.165 μM and 0.011μM, respectively. The concentration levels of Diazepam at 5 and 60 minwere 5.481 μM and 0.176 μM, respectively.

The present invention has been described with reference to variousspecific and preferred embodiments and techniques. However, it should beunderstood that many variations and modifications may be made whileremaining within the spirit and scope of the present invention.

Unless defined otherwise, all scientific and technical terms used hereinhave the same meaning as commonly understood by one of skill in the artto which this invention belongs. Although methods and materials similaror equivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed above. Additionally, the materials, methods, and examples areillustrative only and not intended to be limiting. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control.

1. A compound of formula (I),

or at least one pharmaceutically acceptable salt thereof, wherein R₁ isselected from

R₅₀, R_(50a), and R_(50b) are independently selected from —H, halogen,—OH, —SH, —CN, —C(O)-alkyl, —NR₇R₈, —NO₂, —S(O)₀₋₂-alkyl, alkyl, alkoxy,—O-benzyl (optionally substituted with at least one group independentlyselected from —H, —OH, and alkyl), —C(O)—NR₇R₈, alkyloxy,alkoxyalkoxyalkoxy, and cycloalkyl; wherein the alkyl, alkoxy, andcycloalkyl groups within R₅₀, R_(50a), and R_(50b) are optionallysubstituted with at least one group independently selected from alkyl,halogen, OH, NR₅R₆, CN, haloalkoxy, NR₇R₈, and alkoxy; R₅ and R₆ areindependently selected from —H and alkyl, or R₅ and R₆, and the nitrogento which they are attached, form a 5 or 6 membered heterocycloalkylring; and R₇ and R₈ are independently selected from —H, alkyl optionallysubstituted with at least one group independently selected from —OH,—NH₂, and halogen, -cycloalkyl, and -alkyl-O-alkyl; R₂ is selected from—H, -alkyl optionally substituted with at least one group independentlyselected from R₂₀₀, —OH, —O-alkyl optionally substituted with at leastone group independently selected from R₂₀₀, —C(O)—N(R₃₁₅)(R₃₂₀), whereinR₃₁₅ and R₃₂₀ are each independently selected from —H, alkyl, and aryl,—NH—R₄₀₀, and —R₄₀₀ R₄₀₀ is

wherein R₄₀₅ is selected from —H, —N(R₅₁₅)₂ and O-alkyl; R₅₁₅ isindependently selected from —H, -alkyl, and -aryl; R_(c) is selectedfrom formula

 wherein, A₁ and A₂ are independently selected from —(CH₂)₀₋₂—,—CH(R₂₀₀)—, —C(R₂₀₀)₂—, —C(═N—R₂₃₀)—, —C(═CH—R₂₃₀)—, —C(═N—C(O)—R₂₃₀)—,and —C(═CH—C(O)—R₂₃₀)—; A₃, A₄, and A₅, are independently selected from—CH₂—, —CH(R₂₀₀)—, —C(R₂₀₀)₂—, —C(O)—, —C(═N—R₂₃₀)—, —C(═N—NH(alkyl))-,—C(═N—N(alkyl)(alkyl))-, —C(═N—O—(CH₂)₁₋₄—OH)—, —C(═CH—R₂₃₀),—C(═N—C(O)—R₂₃₀)—, and —C(═CH—C(O)—R₂₃₀)—; R₂₃₀ is independentlyselected from —H, —OH, R₂₁₅ (optionally substituted with —OH, —NH₂,—C(O)H, and —CN), alkyl, cycloalkyl, alkoxy, -alkyl-OH, -alkyl-NH₂,-alkyl-C(O)H, —O—R₂₁₅ (optionally substituted with —OH, —NH₂, —C(O)H,and —CN), —O-alkyl, —O-alkyl-OH, —O-alkyl-NH₂, —O-alkyl-C(O)H, —NH₂,—NHR₂₁₅, —N(R₂₁₅)₂, —NR₂₃₅R₂₄₀, and —CN; wherein at least one carbon ofthe alkyl or cycloalkyl within R₂₃₀ is optionally independently replacedwith —C(O)— or a heteroatom; wherein the cycloalkyl within formulae(Illd) may optionally contain at least one double bond; wherein informulae (Illd), only one of A₁, A₂, A₃, A₄, or A₅ is selected from—C(═N—R₂₃₀)—, —C(═N—NH(alkyl))-, —C(═N—N(alkyl)(alkyl))-,C(═N—O—(CH₂)₁₋₄—OH)—, —C(═CH—R₂₃₀)-r-C(═N—C(O)—R₂₃₀)—, and—C(═CH—C(O)—R₂₃₀)—; wherein each aryl group attached directly orindirectly to R_(C) is optionally substituted with at least one groupindependently selected from R₂₀₀; wherein each cycloalkyl or attacheddirectly or indirectly to R_(C) is optionally substituted with at leastone group independently selected from R₂₁₀; and R_(x) is selected fromaryl, and cycloalkyl; wherein each aryl group of R_(x) is optionallysubstituted with at least one group independently selected from R₂₀₀;wherein each cycloalkyl of R_(x) is optionally substituted with at leastone group independently selected from R₂₁₀, and R₂₀₀ at each occurrenceis independently selected from -alkyl optionally substituted with atleast one group independently selected from R₂₀₅, —(CH₂)₀₋₄—C(O)H,—(CO)₁R₂₁₅, —(CO)₁R₂₂₀, —(CH₂)₀₋₄—(CO)₀₋₁—NR₂₂₀R₂₂₅,—(CH₂)₀₋₄—(CO)₀₋₁—NH(R₂₁₅), —(CH₂)₀₋₄—C(O)-alkyl—(CH₂)₀₋₄—(CO)₀₋₁-cycloalkyl, —(CH₂)₁₋₄—(CO)₀₋₁-heterocycloalkyl,—(CH₂)₀₋₄—(CO)₀₋₁-aryl, —(CH₂)₁₋₄—(CO)₀₋₁-heteroaryl,—(CH₂)₀₋₄—C(O)—O—R₂₁₅, —(CH₂)₀₋₄—SO₂—NR₂₂₀R₂₂₅, —(CH₂)₀₋₄—S(O)₀₋₂-alkyl,—(CH₂)₀₋₄—S(O)₀₋₂-cycloalkyl, —(CH₂)₀₋₄—N(H or R₂₁₅)—C(O)—O—R₂₁₅,—(CH₂)₀₋₄—N(H or R₂₁₅)—SO₂—R₂₂₀, —(CH₂)₀₋₄—N(H or R₂₁₅)—C(O)—N(R₂₁₅)₂,—(CH₂)₀₋₄—N(H or R₂₁₅)—C(O)—R₂₂₀, —(CH₂)₀₋₄—C(O)-alkyl—(CH₂)₀₋₄—O—(R₂₁₅), —(CH₂)₀₋₄—S—(R₂₁₅), and —(CH₂)₀₋₄—O-alkyl optionallysubstituted with at least one halogen; wherein each aryl and heteroarylgroup included within R₂₀₀ is optionally substituted with at least onegroup independently selected from R₂₀₅, R₂₁₀, and alkyl (optionallysubstituted with at least one group independently selected from R₂₀₅ andR₂₁₀); wherein each cycloalkyl or heterocycloalkyl group included withinR₂₀₀ is optionally substituted with at least one group independentlyselected from R₂₁₀; R₂₀₅ at each occurrence is independently selectedfrom -alkyl, -haloalkoxy, —(CH₂)₀₋₃-cycloalkyl, -halogen, —(CH₂)₁₋₆—OH,—O-aryl, —OH, —SH, —(CH₂)₀₋₄—(O)H, —(CH₂)₀₋₆—CN,—(CH₂)₀₋₆—C(O)—NR₂₃₅R₂₄₀, —(CH₂)₀₋₆—C(O)—NR₂₃₅, —(CH₂)₀₋₄—N(H orR₂₁₅)—SO₂—R₂₃₅, —OCF₃, —CF₃, -alkoxy, -alkoxycarbonyl, and —NR₂₃₅R₂₄₀;R₂₁₀ at each occurrence is independently selected from —(CH₂)₀₋₄—OH,—(CH₂)₀₋₄—CN, —(CH₂)₀₋₄—C(O)H, -alkyl optionally substituted with atleast one group independently selected from R₂₀₅, -alkanoyl, —S-alkyl;—S(O)₂-alkyl, -halogen, -alkoxy, —(CH₂)₀₋₄—NR₂₃₅R₂₄₀,—(CH₂)₀₋₄—NR₂₃₅(alkoxy), —(CH₂)₀₋₄—S—(R₂₁₅), —(CH₂)₀₋₄—NR₂₃₅—C(O)H,—(CH₂)₀₋₄—NR₂₃₅—C(O)-(alkoxy), —(CH₂)₀₋₄—NR₂₃₅—C(O)—R₂₄₀, —C(O)—NHR₂₁₅,—C(O)-alkyl, —C(O)—NR₂₃₅R₂₄₀, and R₂₁₅ at each occurrence isindependently selected from -alkyl, —(CH₂)₀₋₂-aryl,—(CH₂)₀₋₂-cycloalkyl, —(CH₂)₀₋₂-heteroaryl, —(CH₂)₀₋₂-heterocycloalkyl,and —CO₂—CH₂-aryl; wherein the aryl group included within R₂₁₅ isoptionally substituted with at least one group independently selectedfrom R₂₀₅ and R₂₁₀, and wherein the heterocycloalkyl and heteroarylgroups included within R₂₁₅ are optionally substituted with at least onegroup independently selected from R₂₁₀; R₂₂₀ and R₂₂₅ at each occurrenceare independently selected from —H, -alkyl, —(CH₂)₀₋₄—C(O)H,-alkylhydroxyl, -alkoxycarbonyl, -alkylamino, —S(O)₂-alkyl, -alkanoyloptionally substituted with at least one halogen, —C(O)—NH₂,—C(O)—NH(alkyl), —C(O)—N(alkyl)(alkyl), -haloalkyl,—(CH₂)₀₋₂-cycloalkyl, -(alkyl)-O-(alkyl), -aryl, -heteroaryl, and-heterocycloalkyl; wherein the aryl, heteroaryl, cycloalkyl, andheterocycloalkyl groups included within R₂₂₀ and R₂₂₅ are eachoptionally substituted with at least one group independently selectedfrom R₂₇₀; R₂₇₀ at each occurrence is independently selected from —R₂₀₅,-alkyl optionally substituted with at least one group independentlyselected from R₂₀₅, -aryl, -halogen, -alkoxy, -haloalkoxy, —NR₂₃₅R₂₄₀,—OH, —CN, -cycloalkyl optionally substituted with at least one groupindependently selected from R₂₀₅, —C(O)-alkyl, —S(O)₂—NR₂₃₅R₂₄₀,—C(O)—NR₂₃₅R₂₄₀, —S(O)₂-alkyl, and —(CH₂)₀₋₄—C(O)H; R₂₃₅ and R₂₄₀ ateach occurrence are independently selected from —H, —OH, —CF₃, —OCH₃,—NHCH₃, —N(CH₃)₂, —(CH₂)₀₋₄—C(O)(H or alkyl), -alkyl, -alkanoyl,—SO₂-alkyl, and -aryl.
 2. The compound according to claim 1, wherein R₁is optionally substituted with at least one group independently selectedfrom halogen, C₁-C₂ alkyl, C₁-C₂ alkoxy, and —OH.
 3. The compoundaccording to claim 1, wherein R₁ is selected from3-allyloxy-5-fluoro-benzyl, 3-benzyloxy-5-fluoro-benzyl,4-hydroxy-benzyl, 3-hydroxybenzyl, 3,5-difluoro-2-propylamino-benzyl,2-ethylamino-3,5-difluoro-benzyl, 2-hydroxy-5-methyl-benzamide,3-fluoro-5-[2-(2-methoxy-ethoxy)-ethoxy]-benzyl,3-fluoro-5-heptyloxy-benzyl, 3-fluoro-5-hexyloxy-benzyl,4-hydroxybenzyl, 3-hydroxy-benzyl, 3,5-difluoro-2-hydroxy-benzyl,3,5-difluoro-4-hydroxy-benzyl, 3,5-difluoro-benzyl,3-fluoro-4-hydroxy-benzyl, 3-fluoro-5-hydroxy-benzyl, and3-fluoro-benzyl.
 4. The compound according to claim 1, wherein R₂ isselected from hydrogen, 3-4-dioxo-cyclobut-1-enylamino,2-methoxy-3-4-dioxo-cyclobut-1-enylamino, and2-methylamino-3,4-dioxo-cyclobut-1-enylamino.
 5. The compound accordingto claim 1, wherein R_(C) is selected from

wherein A₅ is —C(═N—R₂₃₀) and A₁, A₂, A₃, A₄, R_(x) and R₂₃₀ are definedas in claim
 1. 6. The compound according to claim 5, wherein A₅ isselected from —C(═N—OH)—, —C(═N—O—CH₃)—, —C(═N—O—CH₂CH₃)—,—C(═N—O—CH₂CH₂OH)—, —C(═N—O—CH₂CH₂NH₂)—, —C(═N—NHCH₃)—, and —C(═N—CN)—,and A₁, A₂, A₃, and A₄ are —CH₂—.
 7. The compound according to claim 1,wherein R_(C) is selected from1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-methoxyiminocyclohexyl,1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-(2-amino-ethoxyimino)-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexyl,1-(3-tert-Butyl-phenyl)-4-cyanoiminocyclohexyl, 1-(Acrylic acid methylester)-4-(tert-Butyl-phenyl)-cyclohexane-4-yl,1-(Acrylamide)-4-(tert-Butyl-phenyl)-cyclohexane-4-yl,1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethylidene)-cyclohex-1-yl,1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohex-1-yl,1(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohex-1-yl,4-methoxyimino-1-(3-thiophen-3-yl-phenyl)-cyclohexyl,1-(3-furan-3-ylphenyl)-4-methoxyimino-cyclohexyl,4-methoxyimino-1-[3-(1H-pyrrol-2-yl)-phenyl]cyclohexyl,4-methoxyimino-1-(3-pyridin-4-yl-phenyl)-cyclohexyl,4-methoxyimino-I-(3-pyrimidin-5-yl-phenyl)-cyclohexyl,4-methoxyimino-1-(3-pyrazol-1-yl-phenyl)-cyclohexyl,1-(3-tert-Butylphenyl)-4-methylene-cyclohexyl, and ethyl2-(4-(3tert-butylphenyl)cyclohexylidene)acetate.
 8. The compoundaccording to claim 1, wherein R_(x) is selected from3-(1,1-dimethyl-propyl)-phenyl, 3-(1-ethyl-propyl)-phenyl,3-(1-hydroxy-1-methyl-ethyl)-phenyl, 3-(1-methylcyclopropyl)-phenyl,3-(2,2-dimethyl-propyl)-phenyl, 3-(2-fluoro-benzyl)-phenyl,3-(3-methyl-butyl)-phenyl, 3-(4-fluoro-benzyl)-phenyl,3-(Cyano-dimethyl-methyl)-phenyl, 3-Acetyl-5-tert-butyl-phenyl,3′-Acetylamino-biphenyl-3-yl, 3-Bromo-5-tert-butyl-phenyl,3-Cyano-phenyl, 3-Cyclobutyl-phenyl, 3-Cyclopentyl-phenyl,3-Cyclopropyl-phenyl, 3-ethyl-phenyl, 3-ethynyl-phenyl,3-fluoro-5-(2-hydroxy-1,1-dimethyl-ethyl)-phenyl, 3-isobutyl-phenyl,3-isopropyl-phenyl, 3-pent-4-enyl-phenyl, 3-pentyl-phenyl,3-Phenyl-propionic acid ethyl ester, 3-sec-Butyl-phenyl,3-tert-Butyl-4-chloro-phenyl, 3-tert-Butyl-4-cyano-phenyl,3-tert-Butyl-4-ethyl-phenyl, 3-tert-Butyl-4-methyl-phenyl,3-tert-Butyl-4-trifluoromethyl-phenyl, 3-tert-Butyl-5-chloro-phenyl,3-tert-Butyl-5-cyano-phenyl, 3-tert-Butyl-5-ethyl-phenyl,3-tert-Butyl-5-fluoro-phenyl, 3-tert-Butyl-5-methyl-phenyl,3-tert-Butyl-5-trifluoromethyl-phenyl, 3-tert-Butyl-phen-1-yl,3-tert-Butyl-phenyl, 3-trifluoromethyl-phenyl, and4-Acetyl-3-tert-butyl-phenyl.
 9. The compound according to claim 1,wherein R_(x) is 3-tert-Butyl-phen-1-yl.
 10. The compound according toclaim 1, wherein the formula (I) compound is selected from4-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-(cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[4-(2-Amino-ethoxyimino)-1-(3-tert-butyl-phenyl)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexylamino]-2(3,5-difluoro-benzyl)-3-hydroxy-N-methyl]-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methylcarbamoylmethylene-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,{4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-methylcarbamoyl-butylamino]-cyclohexylidene}-aceticacid methyl ester,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxyethylidene)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-methyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1(3-tert-Butyl-phenyl)-4-ethoxyimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxy-ethoxyimino)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[4-(2-Amino-ethoxyimino)-1-(3-tert-butyl-phenyl)-cyclohexylamino)-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(methyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-(dimethyl-hydrazono)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-cyanoimino-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-[1-(3-tert-Butyl-phenyl)-4-methylcarbamoylmethylene-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,{4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-3-phenylcarbamoyl-butylamino]-cyclohexylidene)-aceticacid methyl ester,4-[1-(3-tert-Butyl-phenyl)-4-(2-hydroxyethylidene)-cyclohexylamino]-2-(3,5-difluoro-benzyl)-3-hydroxy-N-phenyl-butyramide,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[3-(3,5-difluorophenoxy)-2-hydroxy-propylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-4-oxo-butylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-pentylamino]-cyclohexanoneoxime,4-(3-tert-Butyl-phenyl)-4-[4-(3,5-difluoro-phenyl)-2-hydroxy-pentylamino]-cyclohexanoneoxime,3-[3-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]1-(3,5-difluoro-benzyl)-2-hydroxy-propylamino]-4-methylamino-cyclobut-3-ene-1,2-dione,and3-[3-[1-(3-tert-Butyl-phenyl)-4-hydroxyimino-cyclohexylamino]1-(3,5-difluoro-benzyl)-2-hydroxy-propylamino]-4-methoxy-cyclobut-3-ene-1,2-dione.